Methods and compositions for attenuating gene therapy anti-viral transfer vector immune responses

ABSTRACT

Provided herein are methods and related compositions for administering viral transfer vectors and antigen-presenting cell targeted immunosuppressants.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119 of U.S.provisional application 62/047,034, filed Sep. 7, 2014; 62/051,255,filed Sep. 16, 2014; 62/101,841, filed Jan. 9, 2015; 62/047,044, filedSep. 7, 2014, 62/051,258, filed Sep. 16, 2014; 62/101,861, filed Jan. 9,2015; 62/047,054, filed Sep. 7, 2014; 62/051,263, filed Sep. 16, 2014;62/101,872, filed Jan. 9, 2015; 62/047,051, filed Sep. 7, 2014,62/051,267, filed Sep. 16, 2014; and 62/101,882, filed Jan. 9, 2015; theentire contents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to methods and compositions for administeringviral transfer vectors and antigen-presenting cell targetedimmunosuppressants.

SUMMARY OF THE INVENTION

Provided herein are methods and compositions for administering genetherapy viral transfer vectors and antigen-presenting cell targetedimmunosuppressants. The viral transfer vector comprises a gene therapytransgene that may have a therapeutic benefit for any one of thepurposes provided herein in any one of the methods or compositionsprovided herein.

In one aspect is a method comprising establishing an anti-viral transfervector attenuated response in a subject by concomitant administration ofan antigen-presenting cell targeted immunosuppressant and viral transfervector to the subject. In one embodiment, the subject does not havepre-existing immunity against the viral transfer vector.

In one embodiment of any one of the methods provided herein, theanti-viral transfer vector attenuated response is a T cell responseagainst the viral transfer vector, and the method further comprisesadministering the viral transfer vector to the subject without anantigen-presenting cell targeted immunosuppressant prior to theconcomitant administration of the antigen-presenting cell targetedimmunosuppressant and viral transfer vector.

In one embodiment of any one of the methods provided herein, theconcomitant administration of the antigen-presenting cell targetedimmunosuppressant and viral transfer vector is repeated, concomitantadministration of the antigen-presenting cell targeted immunosuppressantand viral transfer vector.

In another aspect is a method comprising establishing an anti-viraltransfer vector attenuated response in a subject by concomitantadministration of an antigen-presenting cell targeted immunosuppressantand viral transfer vector to the subject, and administering to thesubject one or more repeat doses of the viral transfer vector.

In one embodiment of any one of the methods provided herein, theanti-viral transfer vector attenuated response is a T cell responseagainst the viral transfer vector, and the method further comprisesadministering the viral transfer vector to the subject without anantigen-presenting cell targeted immunosuppressant prior to both theconcomitant administration of the antigen-presenting cell targetedimmunosuppressant and viral transfer vector and the one or more repeatdoses of the viral transfer vector.

In one embodiment of any one of the methods provided herein, the methodfurther comprises providing or obtaining an antigen-presenting celltargeted immunosuppressant alone or in combination with a viral transfervector.

In another aspect is a method comprising attenuating an anti-viraltransfer vector response, wherein the anti-viral transfer vectorresponse is a T cell response, by first administering to a subject aviral transfer vector without an antigen-presenting cell targetedimmunosuppressant, and subsequently concomitantly administering theviral transfer vector and an antigen-presenting cell targetedimmunosuppressant to the subject.

In one embodiment of any one of the methods provided, the method furthercomprises administering to the subject one or more repeat doses of theviral transfer vector subsequent to the concomitant administration ofthe viral transfer vector and the antigen-presenting cell targetedimmunosuppressant to the subject.

In another aspect is a method comprising determining a level ofpre-existing immunity to a viral transfer vector in a subject prior toadministration of the viral transfer vector to the subject,concomitantly administering to the subject an antigen-presenting celltargeted immunosuppressant and viral transfer vector, and administeringto the subject a dose of the viral transfer vector.

In one embodiment of any one of the methods provided, the determiningcomprises measuring a level of anti-viral transfer vector antibodies inthe subject prior to administration of the viral transfer vector to thesubject. In another embodiment of any one of the methods provided, thedetermining comprises measuring a level of a T cell response against theviral transfer vector in the subject prior to administration of theviral transfer vector to the subject.

In one embodiment of any one of the methods provided, the method furthercomprises one or more repeat doses of the viral transfer vector.

In one embodiment of any one of the methods provided, the level ofpre-existing immunity is to a viral antigen of the viral transfervector. In one embodiment of any one of the methods provided, the levelof pre-existing immunity is to an antigen of a protein transgeneexpression product of the viral transfer vector.

In another aspect is a method comprising escalating transgene expressionof a viral transfer vector in a subject by repeatedly, concomitantlyadministering to the subject an antigen-presenting cell targetedimmunosuppressant and viral transfer vector.

In one embodiment of any one of the methods provided, the method furthercomprises determining the frequency and dosing of the repeated,concomitant administration of the antigen-presenting cell targetedimmunosuppressant and viral transfer vector that increase the transgeneexpression in a subject.

In another aspect is a method comprising repeatedly, concomitantlyadministering to a subject an antigen-presenting cell targetedimmunosuppressant and viral transfer vector, and selecting one or moredoses of the viral transfer vector to be less than the dose of the viraltransfer vector that would be selected for the subject if the subjectwere expected to develop anti-viral transfer vector immune responses dueto the repeated administration of the viral transfer vector.

In another aspect is a method comprising inducing an entity to purchaseor obtain an antigen-presenting cell targeted immunosuppressant alone orin combination with a viral transfer vector by communicating to theentity that concomitant administration of the antigen-presenting celltargeted immunosuppressant and viral transfer vector results in ananti-viral transfer vector attenuated response in a subject.

In another aspect is a method comprising inducing an entity to purchaseor obtain an antigen-presenting cell targeted immunosuppressant alone orin combination with a viral transfer vector by communicating to theentity that efficacious repeated viral transfer vector dosing ispossible by concomitant administration of the antigen-presenting celltargeted immunosuppressant and viral transfer vector to a subject.

In one embodiment of any one of the methods provided herein, thecommunicating further includes instructions for practicing any one ofthe methods described herein or information describing the benefits ofconcomitant administration of a viral transfer vector with anantigen-presenting cell targeted immunosuppressant.

In one embodiment of any one of the methods provided herein, the methodfurther comprises distributing an antigen-presenting cell targetedimmunosuppressant or a viral transfer vector or both to an entity.

In another aspect is a method comprising determining the frequency anddosing of concomitant administration of an antigen-presenting celltargeted immunosuppressant and viral transfer vector in order togenerate an anti-viral transfer vector attenuated response in a subject.

In one embodiment of any one of the methods provided herein, the methodfurther comprises directing the concomitant administration of theantigen-presenting cell targeted immunosuppressant and viral transfervector to a subject according to the determined frequency and dosing.

In another aspect is a method comprising determining the frequency anddosing of concomitant administration of an antigen-presenting celltargeted immunosuppressant and viral transfer vector in combination withone or more repeat doses of the viral transfer vector in order togenerate an anti-viral transfer vector attenuated response in a subject.

In one embodiment of any one of the methods provided herein, the methodfurther comprises directing both the concomitant administration of theantigen-presenting cell targeted immunosuppressant and viral transfervector and administration of the one or more repeat doses of the viraltransfer vector to a subject according to the determined frequency anddosing.

In one embodiment of any one of the methods provided herein, the methodfurther comprises directing the administration of a dose of the viraltransfer vector to the subject prior to both the concomitantadministration of the antigen-presenting cell targeted immunosuppressantand viral transfer vector and administration of the one or more repeatdoses of the viral transfer vector to the subject.

In one embodiment of any one of the methods provided herein, the subjectis one to which the viral transfer vector has not been previouslyadministered.

In one embodiment of any one of the methods provided herein, the subjectis one to which the viral transfer vector has been previouslyadministered no more than once.

In one embodiment of any one of the methods provided, the amount of theviral transfer vector in the repeat dose(s) is at least equal to theamount of the viral transfer vector in a prior dose. In one embodimentof any one of the methods provided, the amount of the viral transfervector in the repeat dose(s) is less than the amount of the viraltransfer vector in a prior dose.

In one embodiment of any one of the methods provided, theantigen-presenting cell targeted immunosuppressant is also administeredto the subject concomitantly with the one or more repeat doses of theviral transfer vector. In one embodiment of any one of the methodsprovided, the antigen-presenting cell targeted immunosuppressant is notalso administered to the subject concomitantly with at least one of theone or more repeat doses of the viral transfer vector.

In one embodiment of any one of the methods provided, the subject doesnot have pre-existing immunity against the viral transfer vector.

In one embodiment of any one of the methods provided, the concomitantadministration is simultaneous administration.

In one embodiment of any one of the methods provided, the method furthercomprises determining a level of pre-existing immunity to the viraltransfer vector in the subject.

In one embodiment of any one of the methods provided herein, the viraltransfer vector is a retroviral transfer vector, an adenoviral transfervector, a lentiviral transfer vector or an adeno-associated viraltransfer vector.

In one embodiment of any one of the methods provided herein, the viraltransfer vector is an adenoviral transfer vector, and the adenoviraltransfer vector is a subgroup A, subgroup B, subgroup C, subgroup D,subgroup E, or subgroup F adenoviral transfer vector.

In one embodiment of any one of the methods provided herein, the viraltransfer vector is a lentiviral transfer vector, and the lentiviraltransfer vector is an HIV, SIV, FIV, EIAV or ovine lentiviral vector.

In one embodiment of any one of the methods provided herein, the viraltransfer vector is an adeno-associated viral transfer vector, and theadeno-associated viral transfer vector is an AAV1, AAV2, AAV5, AAV6,AAV6.2, AAV7, AAV8, AAV9, AAV10 or AAV11 adeno-associated viral transfervector.

In one embodiment of any one of the methods provided herein, the viraltransfer vector is a chimeric viral transfer vector. In one embodimentof any one of the methods provided herein, the chimeric viral transfervector is an AAV-adenoviral transfer vector.

In one embodiment of any one of the methods provided herein, the genetherapy transgene encodes a therapeutic protein. In one embodiment ofany one of the methods provided herein, the therapeutic protein is a/aninfusible or injectable therapeutic protein, enzyme, enzyme cofactor,hormone, blood or blood coagulation factor, cytokine, interferon orgrowth factor. In one embodiment of any one of the methods providedherein, the therapeutic protein is a protein associated with a disorderof lipid and sphingolipid degradation, disorder of mucopolysaccharidedegradation, disorder of glycoprotein degradation, lysosomal storagedisorder or a leukodystrophy.

In one embodiment of any one of the methods provided herein, theantigen-presenting cell targeted immunosuppressant comprises anerythrocyte-binding therapeutic. In one embodiment of any one of themethods provided herein, the erythrocyte-binding therapeutic comprisesERY1, ERY19, ERY59, ERY64, ERY123, ERY141 and ERY162. In one embodimentof any one of the methods provided herein, the erythrocyte-bindingtherapeutic further comprises a viral transfer vector antigen. In oneembodiment of any one of the methods provided herein, the viral transfervector antigen is a viral antigen.

In one embodiment of any one of the methods provided herein, theantigen-presenting cell targeted immunosuppressant comprises anegatively-charged particle. In one embodiment of any one of the methodsprovided herein, the negatively-charged particle is a polystyrene, PLGA,or diamond particle. In one embodiment of any one of the methodsprovided herein, the zeta potential of the particle is negative. In oneembodiment of any one of the methods provided herein, the zeta potentialof the particle is less than −50 mV. In one embodiment of any one of themethods provided herein, the zeta potential of the particle is less than−100 mV.

In one embodiment of any one of the methods provided herein, theantigen-presenting cell targeted immunosuppressant comprises anapoptotic-body mimic and one or more viral transfer vector antigens. Inone embodiment of any one of the methods provided herein, theapoptotic-body mimic is a particle that comprises the one or more viraltransfer vector antigens. In one embodiment of any one of the methodsprovided herein, the one or more viral transfer vector antigens compriseone or more viral antigens. In one embodiment of any one of the methodsprovided herein, the particle may also comprise an apoptotic signalingmolecule. In one embodiment of any one of the methods provided herein,the particle comprises a polyglycolic acid polymer (PGA), polylacticacid polymer (PLA), polysebacic acid polymer (PSA),poly(lactic-co-glycolic) acid copolymer (PLGA), poly(lactic-co-sebacic)acid copolymer (PLSA), poly(glycolic-co-sebacic) acid copolymer (PGSA),polylactide co-glycolide (PLG), or polyethylene glycol (PEG). In oneembodiment of any one of the methods provided herein, the averagediameter of the particle is between 0.1 and 5 μm, between 0.1 and 4 μm,between 0.1 and 3 μm, between 0.1 and 2 μm, between 0.1 and 1 μm orbetween 0.1 and 500 nm.

In one embodiment of any one of the methods provided herein, theantigen-presenting cell targeted immunosuppressant comprises syntheticnanocarriers comprising an immunosuppressant. In one embodiment of anyone of the methods provided herein, the synthetic nanocarriers furthercomprise a viral transfer vector antigen. In one embodiment of any oneof the methods provided herein, the viral transfer vector antigen is aviral antigen. In one embodiment of any one of the methods providedherein, the immunosuppressant and/or the antigen, if present, are/isencapsulated in the synthetic nanocarriers.

In one embodiment of any one of the methods provided herein, thesynthetic nanocarriers comprise lipid nanoparticles, polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, buckyballs, nanowires, virus-like particles or peptide orprotein particles. In one embodiment of any one of the methods providedherein, the synthetic nanocarriers comprise polymeric nanoparticles. Inone embodiment of any one of the methods provided herein, the polymericnanoparticles comprise a polymer that is a non-methoxy-terminated,pluronic polymer. In one embodiment of any one of the methods providedherein, the polymeric nanoparticles comprise a polyester, polyesterattached to a polyether, polyamino acid, polycarbonate, polyacetal,polyketal, polysaccharide, polyethyloxazoline or polyethyleneimine. Inone embodiment of any one of the methods provided herein, the polyestercomprises a poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid) or polycaprolactone. In one embodiment ofany one of the methods provided herein, the polymeric nanoparticlescomprise a polyester and a polyester attached to a polyether. In oneembodiment of any one of the methods provided herein, the polyethercomprises polyethylene glycol or polypropylene glycol.

In one embodiment of any one of the methods provided herein, the mean ofa particle size distribution obtained using dynamic light scattering ofa population of the synthetic nanocarriers is a diameter greater than110 nm. In one embodiment of any one of the methods provided herein, thediameter is greater than 150 nm. In one embodiment of any one of themethods provided herein, the diameter is greater than 200 nm. In oneembodiment of any one of the methods provided herein, the diameter isgreater than 250 nm. In one embodiment of any one of the methodsprovided herein, the diameter is less than 5 μm. In one embodiment ofany one of the methods provided herein, the diameter is less than 4 μm.In one embodiment of any one of the methods provided herein, thediameter is less than 3 μm. In one embodiment of any one of the methodsprovided herein, the diameter is less than 2 μm. In one embodiment ofany one of the methods provided herein, the diameter is less than 1 μm.In one embodiment of any one of the methods provided herein, thediameter is less than 500 nm. In one embodiment of any one of themethods provided herein, the diameter is less than 450 nm. In oneembodiment of any one of the methods provided herein, the diameter isless than 400 nm. In one embodiment of any one of the methods providedherein, the diameter is less than 350 nm. In one embodiment of any oneof the methods provided herein, the diameter is less than 300 nm.

In one embodiment of any one of the methods provided herein, the load ofimmunosuppressant comprised in the synthetic nanocarriers, on averageacross the synthetic nanocarriers, is between 0.1% and 50%(weight/weight). In one embodiment of any one of the methods providedherein, the load is between 0.1% and 25%. In one embodiment of any oneof the methods provided herein, the load is between 1% and 25%. In oneembodiment of any one of the methods provided herein, the load isbetween 2% and 25%.

In one embodiment of any one of the methods provided herein, theimmunosuppressant is an inhibitor of the NF-kB pathway. In oneembodiment of any one of the methods provided herein, theimmunosuppressant is rapamycin.

In one embodiment of any one of the methods provided herein, an aspectratio of a population of the synthetic nanocarriers is greater than 1:1,1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.

In one embodiment of any one of the methods provided herein, the methodfurther comprises performing the method according to a protocol thatattenuates an anti-viral transfer vector response, such as an antibody,T cell or B cell response, escalates transgene expression or thatestablishes an anti-viral transfer vector response. In one embodiment ofany one of the methods provided herein, the method further comprisesdetermining a protocol that attenuates an anti-viral transfer vectorresponse, such as an antibody, T cell or B cell response, escalatestransgene expression or that establishes an anti-viral transfer vectorresponse.

In another embodiment of any one of the methods provided, the methodfurther comprises assessing an antibody immune response against theviral transfer vector prior to, during or subsequent to theadministering to the subject.

In another aspect a method or composition as described in any one of theExamples is provided.

In another aspect, any one of the compositions is for use in any one ofthe methods provided.

In another aspect, any one of the methods is for use in treating any oneof the disease or disorders described herein. In another aspect, any oneof the methods is for use in attenuating an anti-viral transfer vectorresponse, establishing an attenuated anti-viral transfer vectorresponse, escalating transgene expression or for repeated administrationof a viral transfer vector.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. shows GFP expression in livers of mice injected with AAV with orwithout synthetic nanocarriers comprising rapamycin at prime or boost.All cells in suspension have been analyzed for GFP expression with theexception of high side-scatter debris (2-3% of total, a by-product ofcollagenase treatment) excluded by the first ‘clean’ gate. All theremaining cells were gated for relative GFP strength (FL-1 channel).Numbers shown represent the percentage of GFP-positive cells of thetotal parent population.

FIG. 2. shows GFP expression in livers of AAV-injected mice as afunction of boost with or without synthetic nanocarriers comprisingrapamycin. Data presented are the same as in FIG. 1, but are groupedaccording to whether AAV boost employed co-administration with thesynthetic nanocarriers comprising rapamycin or not (unboosted samplesfrom gr. 5 and 6 are also shown as a separate ‘supergroup’).

FIG. 3. demonstrates the GFP^(high) cell share in livers of animalsinjected with AAV with or without synthetic nanocarriers comprisingrapamycin. GFP-positive cells (as presented in FIG. 1) were gated andthen a population with an average GFP fluorescence intensity of 10 timeshigher than average in the parent population was gated again. Numberspresented are percentage from the parent GFP-positive population as seenin FIG. 1.

FIG. 4. shows results from an experiment where mice were bled at d14after receiving a single AAV-GFP inoculation with or withoutco-administration of synthetic nanocarriers comprising rapamycin andtheir sera assayed for antibodies against AAV. Top ODs for 1:40 serumdilutions are shown for all mice. Background normal mouse serum had anOD of 0.227.

FIG. 5. shows results from an experiment where mice were bled at days14, 21 and 33 after receiving a single AAV-GFP inoculation with orwithout co-administration of synthetic nanocarriers comprising rapamycin(i.v.) and their sera assayed for antibodies against AAV. Top ODs for1:40 serum dilutions are shown for all mice. Background normal mouseserum activity is shown. Statistical significance is calculated usingtwo-way ANOVA.

FIG. 6. shows results from an experiment where mice were injected withAAV-GFP at days 0 and 21 with or without co-administration of syntheticnanocarriers comprising rapamycin (i.v.) at either or both injections,then bled at days 14 and 33 and their sera assayed for antibodiesagainst AAV. Top ODs for 1:40 serum dilutions are shown for all mice.Background normal mouse serum activity is shown. Statisticalsignificance is calculated using two-way ANOVA.

FIG. 7. provides data that are the same as in FIG. 6 with the readingsfor individual mice shown. Two mice in the group treated with syntheticnanocarriers comprising rapamycin only at boost immunization (d21) didnot show detectable antibodies at day 33 despite being positive at d14(solid arrows). One of five mice in both groups treated with syntheticnanocarriers comprising rapamycin at the prime had a detectable antibodylevel at d33 (dashed arrows) with the mouse from the group treated withsynthetic nanocarriers comprising rapamycin at both prime and boosthaving a lower antibody level (open diamonds).

FIG. 8. shows results from an experiment where mice mice were bled atd14 after receiving a single AAV-GFP inoculation with or withoutco-administration of synthetic nanocarriers comprising rapamycin andtheir sera assayed for antibodies against AAV. Top ODs for 1:40 serumdilutions are shown for all mice. Background normal mouse serum had anOD of 0.227. N=15 mice per group.

FIG. 9. shows results from an experiment where mice were bled at days14, 21 and 33 after receiving a single AAV-GFP inoculation with orwithout co-administration of synthetic nanocarriers comprising rapamycin(i.v.) and their sera assayed for antibodies against AAV. Top ODs for1:40 serum dilutions are shown for all mice. Background normal mouseserum levels are shown. Statistical significance is calculated usingtwo-way ANOVA. N=15 mice/group at day 14 and 5 mice/group at days 21 and33.

FIG. 10 shows results from an experiment where mice were injected withAAV8-GFP at days 0 and 21 with or without co-administration of syntheticnanocarriers comprising rapamycin (i.v.) at one or both injections, asindicated, and then bled at days 14 and 33. Sera were assayed forantibodies against AAV8 by ELISA. ODs for 1:40 serum dilutions are shownfor all mice. Background level of normal mouse serum is indicated by thedotted line. Statistical significance is calculated using two-way ANOVA.

FIG. 11 shows GFP expression in livers of mice injected with AAV with orwithout synthetic nanocarriers comprising rapamycin at prime or boost.All cells in suspension have been analyzed for GFP expression with theexception of high side-scatter debris (2-3% of total, a by-product ofcollagenase treatment) excluded by the first ‘clean’ gate. All theremaining cells were gated for relative GFP strength (FL-1 channel).Numbers shown represent the percentage of GFP-positive cells of thetotal parent population.

FIG. 12 shows RFP expression in livers of mice injected with AAV with orwithout synthetic nanocarriers comprising rapamycin at prime and/orboost. All cells in suspension have been analyzed for RFP expressionwith the exception of high side-scatter debris. Numbers shown representthe percentage of RFP-positive cells of the total parent population ofliver cells.

FIG. 13 shows cytotoxic activity in mice immunized with AAV-GFP alone orin combination with synthetic nanocarriers comprising rapamycin. Animalswere injected with AAV8-GFP (i.v.) on days 0 and 21 with or withoutsynthetic nanocarriers comprising rapamycin. Target cells pulsed with acombination of dominant cytotoxic peptides from AAV capsid protein andthe GFP transgene were administered at 7 days after the last injection(day 28) and their viability measured 18 hours later and compared tothat of non-peptide pulsed control cells.

FIG. 14 shows AAV-specific IFN-γ production in mice immunized withAAV-GFP alone or in combination with synthetic nanocarriers comprisingrapamycin. Animals were injected with AAV-GFP (i.v.) on days 0 and 17with or without NCS. Splenocytes were isolated on day 25 and incubatedin vitro with dominant MHC class I-binding peptide from AAV capsidprotein for 7 days and then assayed by ELISpot with the same peptide.Each sample was run in duplicate and presented with backgroundsubtracted.

FIG. 15 shows GFP-specific IFN-γ production in mice immunized withAAV-GFP alone or in combination with synthetic nanocarriers comprisingrapamycin. Animals were injected (i.v.) with AAV8-GFP on days 0 and 17with or without synthetic nanocarriers comprising rapamycin. Splenocyteswere isolated and incubated in vitro with MHC class I-binding peptidefrom GFP for 7 days and then assayed by ELISpot with the same peptide.Each sample was run in duplicate and presented with backgroundsubtracted.

FIG. 16 shows the design for an experiment.

FIG. 17 shows results from an experiment where mice were injected withrAAV2/8-luciferase on day 0 with or without co-administration ofsynthetic nanocarriers carrying 100 μg of rapamycin (i.v.) and thenchallenged with an i.v. injection of AAV-hFIX on day 14. Sera wascollected at various time points, as indicated, and assayed forantibodies against AAV8 (left) and for the levels of human factor IXprotein (right).

FIG. 18 shows the experimental design for an experiment.

FIG. 19 shows results from an experiment where male C57BL/6 mice wereinjected (i.v.) with rAAV2/8-luciferase concomitantly with syntheticnanocarriers carrying 100 μg of rapamycin on day 0 and then injectedwith rAAV2/8-hFIX concomitantly with synthetic nanocarriers carrying 100μg of rapamycin on day 21. Control animals were treated similarly butwith empty nanocarriers instead of synthetic nanocarriers comprisingrapamycin. Sera were collected at various time points, as indicated, andassayed by ELISA for antibodies against AAV (left) and for levels ofhuman FIX protein (right). AAV2/8-FIX vector copy number in the liver(middle) was determined by PCR.

FIG. 20 shows the experimental design for an experiment.

FIG. 21 provides results that showed that concomitant i.v.administration of synthetic nanocarriers carrying rapamycin with anrAAV2/8 vector (AAV2/8-Luc) on day 0 did not have a profound impact onthe antibody response to an AAV5 vector (AAV5-hFIX) administered on day21. In contrast, the results also showed that mice concomitantly treatedwith synthetic nanocarriers comprising rapamycin and rAAV2/8-Luc on day0 showed a robust response to immunization with recombinant hFIX proteinin complete Freund's adjuvant (CFA) on day 21.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified materials or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting of the use of alternativeterminology to describe the present invention.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyfor all purposes. Such incorporation by reference is not intended to bean admission that any of the incorporated publications, patents andpatent applications cited herein constitute prior art.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a polymer”includes a mixture of two or more such molecules or a mixture ofdiffering molecular weights of a single polymer species, reference to “asynthetic nanocarrier” includes a mixture of two or more such syntheticnanocarriers or a plurality of such synthetic nanocarriers, reference to“a DNA molecule” includes a mixture of two or more such DNA molecules ora plurality of such DNA molecules, reference to “an immunosuppressant”includes a mixture of two or more such immunosuppressant molecules or aplurality of such immunosuppressant molecules, and the like.

As used herein, the term “comprise” or variations thereof such as“comprises” or “comprising” are to be read to indicate the inclusion ofany recited integer (e.g. a feature, element, characteristic, property,method/process step or limitation) or group of integers (e.g. features,elements, characteristics, properties, method/process steps orlimitations) but not the exclusion of any other integer or group ofintegers. Thus, as used herein, the term “comprising” is inclusive anddoes not exclude additional, unrecited integers or method/process steps.

In embodiments of any of the compositions and methods provided herein,“comprising” may be replaced with “consisting essentially of” or“consisting of”. The phrase “consisting essentially of” is used hereinto require the specified integer(s) or steps as well as those which donot materially affect the character or function of the claimedinvention. As used herein, the term “consisting” is used to indicate thepresence of the recited integer (e.g. a feature, element,characteristic, property, method/process step or limitation) or group ofintegers (e.g. features, elements, characteristics, properties,method/process steps or limitations) alone.

A. INTRODUCTION

Anti-viral transfer vectors are promising therapeutics for a variety ofapplications. Viral transfer vectors, therefore, may comprise transgenesthat encode any protein that may have a beneficial therapeutic purpose.Unfortunately, the promise of these therapeutics has not yet beenrealized in the art in a large part due to cellular and humoral immuneresponses against the viral transfer vector. These immune responsesinclude antibody, B cell and T cell responses and can be specific toviral antigens of the viral transfer vector, such as viral capsid orcoat proteins or peptides thereof.

Currently, many possible patients harbor some level of pre-existingimmunity against the viruses on which viral transfer vectors are based.In fact, antibodies against viral antigens, such as antibodies againstadeno-associated viruses, are highly prevalent in the human population.In addition, even if the level of pre-existing immunity is low, forexample due to the low immunogenicity of the viral transfer vector, suchlow levels may still prevent successful transduction (e.g., Jeune, etal., Human Gene Therapy Methods, 24:59-67 (2013)). Thus, even low levelsof pre-existing immunity may hinder the use of a specific viral transfervector and may require a clinician to choose a viral transfer vectorbased on a virus of a different serotype, that may not be asefficacious, or even opt for a different type of therapy if anotherviral transfer vector therapy is not available.

Additionally, viral vectors, such as adeno-associated vectors, can behighly immunogenic and elicit humoral and cell-mediated immunity thatcan compromise efficacy, particularly with respect to re-administration.In fact, cellular and humoral immune responses against a viral transfervector can develop after a single administration of the viral transfervector. After viral transfer vector administration, neutralizingantibody titers can increase and remain high for several years and canreduce the effectiveness of readministration of the viral transfervector, as repeated administration of a viral transfer vector generallyresults in enhanced undesired immune responses. In addition, viraltransfer vector-specific CD8+ T cells may arise that eliminatetransduced cells expressing a desired transgene product, such as, forexample, on reexposure to a viral antigen, such as a capsid protein.Indeed, it has been shown that AAV capsid antigen triggeredimmune-mediated destruction of hepatocytes transduced with an AAV viraltransfer vector (e.g., Manno et al., Nature Medicine, Vol. 12, No. 3,2006). For many therapeutic applications, it is anticipated thatmultiple rounds of administration of viral transfer vectors will beneeded for long-term benefits, and, without the methods and compositionsprovided herein, the ability to do so would be expected to be severelylimited particularly if readministration is needed.

The problems associated with the use of viral transfer vectors fortherapy is further compounded because viral transfer vector antigens canpersist for some time, such as for at least several weeks, after asingle administration (e.g., Nathawani et al., N Engl J Med 365; 25,2011; Nathwani, et al., N Engl J Med 371; 21, 2014). As an example, ithas been found that long-lasting capsid-specific humoral immunitydeveloped in patients that received a single infusion of anadeno-associated virus serotype 8 (AAV8) viral transfer vector (e.g.,Nathwani, et al., N Engl J Med 371; 21, 2014). The persistence ofantigen further hinders the ability to use viral transfer vectorssuccessfully. It is important to evade immune responses against viraltransfer vectors in order for therapy with viral transfer vectors to besuccessful. Prior to this invention, however, there was no way to do soand achieve long-term immune response attenuation without the need forlong-term administration of an immunosuppressant.

The inventors have surprisingly and unexpectedly discovered that theproblems and limitations noted above can be overcome by practicing theinvention disclosed herein. Methods and compositions are provided thatoffer solutions to the aformentioned obstacles to effective use of viraltransfer vectors for treatment. In particular, it has been unexpectedlydiscovered that anti-viral transfer vector immune responses can beattenuated with the methods and related compositions provided herein.The methods and compositions can increase the efficacy of treatment withviral transfer vectors and provide for long-term immune attenuation evenif the administration of the viral transfer vector need be repeated.

The invention will now be described in more detail below.

B. DEFINITIONS

“Administering” or “administration” or “administer” means giving ordispensing a material to a subject in a manner that is pharmacologicallyuseful. The term is intended to include “causing to be administered”.“Causing to be administered” means causing, urging, encouraging, aiding,inducing or directing, directly or indirectly, another party toadminister the material. Any one of the methods provided herein maycomprise or further comprise a step of administering concomitantly anantigen-presenting cell targeted immunosuppressant and a viral transfervector. In some embodiments, the concomitant administration is performedrepeatedly. In still further embodiments, the concomitant administrationis simultaneous administration.

“Amount effective” in the context of a composition or dosage form foradministration to a subject as provided herein refers to an amount ofthe composition or dosage form that produces one or more desired resultsin the subject, for example, the reduction or elimination of an immuneresponse against a viral transfer vector or the generation of ananti-viral transfer vector attenuated response. The amount effective canbe for in vitro or in vivo purposes. For in vivo purposes, the amountcan be one that a clinician would believe may have a clinical benefitfor a subject that may experience undesired immune responses as a resultof administration of a viral transfer vector. In any one of the methodsprovided herein, the composition(s) administered may be in any one ofthe amounts effective as provided herein.

Amounts effective can involve reducing the level of an undesired immuneresponse, although in some embodiments, it involves preventing anundesired immune response altogether. Amounts effective can also involvedelaying the occurrence of an undesired immune response. An amounteffective can also be an amount that results in a desired therapeuticendpoint or a desired therapeutic result. Amounts effective, preferably,result in a tolerogenic immune response in a subject to an antigen, suchas a viral transfer vector antigen. Amounts effective, can alsopreferably result in increased transgene expression (the transgene beingdelivered by the viral transfer vector). This can be determined bymeasuring transgene protein concentrations in various tissues or systemsof interest in the subject. This increased expression may be measuredlocally or systemically. The achievement of any of the foregoing can bemonitored by routine methods.

In some embodiments of any one of the compositions and methods provided,the amount effective is one in which the desired immune response, suchas the reduction or elimination of an immune response against a viraltransfer vector or the generation of an anti-viral transfer vectorattenuated response, persists in the subject for at least 1 week, atleast 2 weeks or at least 1 month. In other embodiments of any one ofthe compositions and methods provided, the amount effective is one whichproduces a measurable desired immune response, such as the reduction orelimination of an immune response against a viral transfer vector or thegeneration of an anti-viral transfer vector attenuated response. In someembodiments, the amount effective is one that produces a measurabledesired immune response (e.g., to a specific viral transfer vectorantigen), for at least 1 week, at least 2 weeks or at least 1 month.

Amounts effective will depend, of course, on the particular subjectbeing treated; the severity of a condition, disease or disorder; theindividual patient parameters including age, physical condition, sizeand weight; the duration of the treatment; the nature of concurrenttherapy (if any); the specific route of administration and like factorswithin the knowledge and expertise of the health practitioner. Thesefactors are well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation.

“Anti-viral transfer vector immune response” or “immune response againsta viral transfer vector” or the like refers to any undesired immuneresponse against a viral transfer vector. In some embodiments, theundesired immune response is an antigen-specific immune response againstthe viral transfer vector or an antigen thereof. In some embodiments,the immune response is specific to a viral antigen of the viral transfervector. In other embodiments, the immune response is specific to aprotein or peptide encoded by the transgene of the viral transfervector. In some embodiments, the immune response is specific to a viralantigen of the viral transfer vector and not to a protein or peptidethat is encoded by the transgene of the viral transfer vector. Theimmune response may be an anti-viral transfer vector antibody response,an anti-viral transfer vector T cell immune response, such as a CD4+ Tcell or CD8+ T cell immune response, or an anti-viral transfer vector Bcell immune response.

An anti-viral transfer vector immune response is said to be an“anti-viral transfer vector attenuated response” when it is in somemanner reduced or eliminated in the subject or as compared to anexpected or measured response in the subject or another subject. In someembodiments, the anti-viral transfer vector attenuated response in asubject comprises a reduced anti-viral transfer vector immune response(such as a T cell, B cell or antibody response) measured using abiological sample obtained from the subject following a concomitantadministration as provided herein as compared to an anti-viral transfervector immune response measured using a biological sample obtained fromanother subject, such as a test subject, following administration tothis other subject of the viral transfer vector without concomitantadministration of the antigen-presenting cell targetedimmunosuppressant. In some embodiments, the biological sample isobtained from the other subject following administration to this othersubject of the viral transfer vector without any administration of theantigen-presenting cell targeted immunosuppressant. In some embodiments,the anti-viral transfer vector attenuated response is a reducedanti-viral transfer vector immune response (such as a T cell, B cell orantibody response) in a biological sample obtained from the subjectfollowing a concomitant administration as provided herein upon asubsequent viral transfer vector in vitro challenge performed on thesubject's biological sample as compared to the anti-viral transfervector immune response detected upon viral transfer vector in vitrochallenge performed on a biological sample obtained from anothersubject, such as a test subject, following administration to this othersubject of the viral transfer vector without concomitant administrationof the antigen-presenting cell targeted immunosuppressant. In someembodiments, the anti-viral transfer vector attenuated response is areduced anti-viral transfer vector immune response (such as a T cell, Bcell or antibody response) in the subject following a concomitantadministration as provided herein upon a subsequent viral transfervector challenge administered to the subject as compared to theanti-viral transfer vector immune response in another subject, such as atest subject, upon a viral transfer vector challenge administered tothis other subject following administration to this other subject of theviral transfer vector without concomitant administration of theantigen-presenting cell targeted immunosuppressant. In some embodiments,the viral transfer vector is administered without any administration ofthe antigen-presenting cell targeted immunosuppressant.

“Antigen” means a B cell antigen or T cell antigen. “Type(s) ofantigens” means molecules that share the same, or substantially thesame, antigenic characteristics. In some embodiments, antigens may beproteins, polypeptides, peptides, lipoproteins, glycolipids,polynucleotides, polysaccharides, etc.

“Antigen-presenting cell targeted immunosuppressant” means an agent thatresults in antigen-presenting cells (APCs) having a tolerogenic effect.Such an immunosuppressant can include immunosuppressants coupled to acarrier that results in delivery to APCs and a tolerogenic effect aswell as agents that by virtue of their form or characteristics canresult in APC tolerogenic effects. Examples of antigen-presenting celltargeted immunosuppressants include, but are not limited to syntheticnanocarriers that comprise an immunosuppressant as described herein;immunosuppressants, as described herein, coupled to antibodies orantigen-binding fragments thereof that target APCs (or other ligand thattargets an APC), erythrocyte-binding therapeutics, as well as particlesthat by virtue of their characteristics lead to APC tolerogenic immuneresponses, etc.

When the antigen-presenting cell targeted immunosuppressant is asynthetic nanocarrier coupled to an immunosuppressant, in someembodiments, the immunosuppressant is an element that is in addition tothe material that makes up the structure of the synthetic nanocarrier.For example, in one embodiment, where the synthetic nanocarrier is madeup of one or more polymers, the immunosuppressant is a compound that isin addition and, in some embodiments, attached to the one or morepolymers. As another example, in one embodiment, where the syntheticnanocarrier is made up of one or more lipids, the immunosuppressant isagain in addition to and, in some embodiments, attached to the one ormore lipids. In embodiments where the antigen-presenting cell targetedimmunosuppressant is a synthetic nanocarrier coupled to animmunosuppressant, and the material of the synthetic nanocarrier alsoresults in a tolerogenic effect, the immunosuppressant is an elementpresent in addition to the material of the synthetic nanocarrier thatresults in a tolerogenic effect.

“Antigen-specific” refers to an immune response that results from thepresence of an antigen of interest or that generates molecules thatspecifically recognize or bind the antigen of interest. Generally, whilesuch responses are measurable against the antigen of interest, theresponses are reduced or negligible in regard to other antigens. Forexample, where the immune response is antigen-specific antibodyproduction, antibodies are produced that selectively bind the antigen ofinterest but not to other antigens. As another example, where the immuneresponse involves the production of CD4+ or CD8+ T cells,antigen-specific CD4+ or CD8+ T cells can bind to an antigen of interestor portion thereof when presented in the context of MHC class I or IIantigens, respectively, by an antigen-presenting cell (APC) or, in caseof CD8+ T cells, by any other cell in which the antigen is produced(e.g., a cell infected with a virus). In the case of immune tolerance,antigen specificity refers to the selective prevention or inhibition ofa specific immune response to a target antigen versus other unrelated orunassociated antigens (e.g. antigens that are temporally or spatiallydislocated from the target antigen).

“Assessing an immune response” refers to any measurement ordetermination of the level, presence or absence, reduction, increase in,etc. of an immune response in vitro or in vivo. Such measurements ordeterminations may be performed on one or more samples obtained from asubject. Such assessing can be performed with any one of the methodsprovided herein or otherwise known in the art. The assessing may beassessing the number or percentage of antibodies or T cells, such asthose specific to a viral transfer vector, such as in a sample from asubject. The assessing also may be assessing any effect related to theimmune response, such as measuring the presence or absence of acytokine, cell phenotype, etc. Any one of the methods provided hereinmay comprise or further comprise a step of assessing an immune responseto a viral transfer vector or antigen thereof. The assessing may be donedirectly or indirectly. The term is intended to include actions thatcause, urge, encourage, aid, induce or direct another party to assess animmune response.

“Attach” or “Attached” or “Couple” or “Coupled” (and the like) means tochemically associate one entity (for example a moiety) with another. Insome embodiments, the attaching is covalent, meaning that the attachmentoccurs in the context of the presence of a covalent bond between the twoentities. In non-covalent embodiments, the non-covalent attaching ismediated by non-covalent interactions including but not limited tocharge interactions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. In embodiments,encapsulation is a form of attaching.

“Average”, as used herein, refers to the arithmetic mean unlessotherwise noted.

“Concomitantly” means administering two or more materials/agents to asubject in a manner that is correlated in time, preferably sufficientlycorrelated in time so as to provide a modulation in an immune response,and even more preferably the two or more materials/agents areadministered in combination. In embodiments, concomitant administrationmay encompass administration of two or more materials/agents within aspecified period of time, preferably within 1 month, more preferablywithin 1 week, still more preferably within 1 day, and even morepreferably within 1 hour. In embodiments, the materials/agents may berepeatedly administered concomitantly; that is concomitantadministration on more than one occasion, such as provided in theExamples.

“Determining” means objectively ascertaining something, such as a fact,relationship or quantity. In some embodiments, whether or not a subjecthas a pre-existing immunity to a viral transfer vector may bedetermined. The term is intended to include “causing to be determined”.“Causing to be determined” means causing, urging, encouraging, aiding,inducing or directing another party to perform a step of determining asprovided herein. In some embodiments, the step of determining may bedetermining whether or not a subject has a pre-existing immunity to aviral transfer vector. Any one of the methods provided herein maycomprise or further comprise a step of determining as described hereinincluding a step of determining whether or not a subject has apre-existing immunity to a viral transfer vector.

“Directing” means influencing, such as taking some action to influence,in some manner the actions of another party, such as causing orcontrolling the acts of the other party in such a manner that theyperform one or more steps as provided herein. In some embodiments, theother party is an agent of the party that is doing the directing. Inother embodiments, the other party is not an agent of the party that isdoing the directing, but the step(s) performed by the other party is/areattributable to or the result of the directing. Accordingly, directingincludes instructing or providing instructions to perform one or moresteps in order to receive a benefit conditioned on the performance ofthe one or more steps.

“Dosage form” means a pharmacologically and/or immunologically activematerial in a medium, carrier, vehicle, or device suitable foradministration to a subject. Any one of the compositions or dosesprovided herein may be in a dosage form.

“Dose” refers to a specific quantity of a pharmacologically and/orimmunologically active material for administration to a subject for agiven time. A “prior dose” refers to an earlier dose of a material. Ingeneral, doses of the antigen-presenting cell targetedimmunosuppressants and/or viral transfer vectors in the methods andcompositions of the invention refer to the amount of theantigen-presenting cell targeted immunosuppressants and/or viraltransfer vectors. Alternatively, the dose can be administered based onthe number of synthetic nanocarriers that provide the desired amount ofantigen-presenting cell targeted immunosuppressant, in instances wherethe antigen-presenting cell targeted immunosuppressant is a syntheticnanocarrier that comprises an immunosuppressant. When dose is used inthe context of a repeated dosing, dose refers to the amount of each ofthe repeated doses, which may be the same or different.

“Encapsulate” means to enclose at least a portion of a substance withina synthetic nanocarrier. In some embodiments, a substance is enclosedcompletely within a synthetic nanocarrier. In other embodiments, most orall of a substance that is encapsulated is not exposed to the localenvironment external to the synthetic nanocarrier. In other embodiments,no more than 50%, 40%, 30%, 20%, 10% or 5% (weight/weight) is exposed tothe local environment. Encapsulation is distinct from absorption, whichplaces most or all of a substance on a surface of a syntheticnanocarrier, and leaves the substance exposed to the local environmentexternal to the synthetic nanocarrier.

“Escalating transgene expression” refers to increasing the level of thetransgene expression product of a viral transfer vector in a subject,the transgene being delivered by the viral transfer vector. In someembodiments, the level of the transgene expression product may bedetermined by measuring transgene protein concentrations in varioustissues or systems of interest in the subject. Alternatively, when thetransgene expression product is a nucleic acid, the level of transgeneexpression may be measured by transgene nucleic acid products.Escalating transgene expression can be determined, for example, bymeasuring the amount of the transgene expression product in a sampleobtained from a subject and comparing it to a prior sample. The samplemay be a tissue sample. In some embodiments, the transgene expressionproduct can be measured using flow cytometry.

“Establishing” or “establish” means to generate an outcome or result orto deduce something, such as a fact or relationship. Which use of thisterm will be apparent based on the context in which it is used. Forgenerating an outcome or result, the establishing may be accomplished ina number of ways, including but not limited to, taking steps toaccomplish the outcome or result. For example, in some embodiments,administration of material(s) as provided herein can generate theoutcome or result. For determining something, such as a fact orrelationship, the establishing may be accomplished by performingexperiments, making projections, etc. For instance, establishing thatadministration of a viral transfer vector is likely to generate ananti-viral transfer vector immune response in a subject may be based onresults of experiments on a subject, including on one or more samplesobtained therefrom. Generally, the likelihood of generating ananti-viral transfer vector immune response in a subject is thelikelihood of generating such a response with the administration (orrepeated administration, in some embodiments) of a viral transfer vectorin the absence of administration of an antigen-presenting cell targetedimmunosuppressant as provided herein. Likewise, establishing that asubject has a pre-existing immunity to a viral transfer vector may alsobe based on the result of experiments on a subject, including on one ormore samples obtained therefrom. In another embodiment, suchestablishing may be determined by assessing an immune response in thesubject. In regard to establishing a dose for administration, a dose ofan antigen-presenting cell targeted immunosuppressant or a viraltransfer vector may be determined by starting with a test dose and usingknown scaling techniques (such as allometric or isometric scaling) todetermine the dose for administration. Such may also be used toestablish a protocol as provided herein. “Establishing” or “establish”comprises “causing to be established.” “Causing to be established” meanscausing, urging, encouraging, aiding, inducing or directing or acting incoordination with an entity for the entity to perform a step ofestablishing as provided herein. In some embodiments of any one of themethods provided herein, the method may comprise or further comprise anyone of the steps of establishing as described herein.

“Frequency” refers to the interval of time at which theantigen-presenting cell targeted immunosuppressant, the viral transfervector or both in combination (such as with concomitant administration)are administered to a subject.

“Gene therapy transgene” refers to a nucleic acid that encodes a proteinand that when introduced into a cell directs the expression of theprotein. Preferably, the protein is a therapeutic protein. In someembodiments of any one of the methods or compositions provided herein,the subject to which the gene therapy transgene is administered by wayof a viral transfer vector has a disease or disorder whereby thesubject's endogenous version of the protein is defective or produced inlimited amounts or not at all. In some embodiments, the encoded proteinhas no human counterpart but is predicted to provide therapeuticallybeneficial effects in the treatment of a disease or disorder.

“Immunosuppressant” means a compound that causes a tolerogenic effect,preferably through its effects on APCs. A tolerogenic effect generallyrefers to the modulation by the APC or other immune cells systemicallyand/or locally, that reduces, inhibits or prevents an undesired immuneresponse to an antigen in a durable fashion. In one embodiment, theimmunosuppressant is one that causes an APC to promote a regulatoryphenotype in one or more immune effector cells. For example, theregulatory phenotype may be characterized by the inhibition of theproduction, induction, stimulation or recruitment of antigen-specificCD4+ T cells or B cells, the inhibition of the production ofantigen-specific antibodies, the production, induction, stimulation orrecruitment of Treg cells (e.g., CD4+CD25highFoxP3+ Treg cells), etc.This may be the result of the conversion of CD4+ T cells or B cells to aregulatory phenotype. This may also be the result of induction of FoxP3in other immune cells, such as CD8+ T cells, macrophages and iNKT cells.In one embodiment, the immunosuppressant is one that affects theresponse of the APC after it processes an antigen. In anotherembodiment, the immunosuppressant is not one that interferes with theprocessing of the antigen. In a further embodiment, theimmunosuppressant is not an apoptotic-signaling molecule. In anotherembodiment, the immunosuppressant is not a phospholipid.

Immunosuppressants include, but are not limited to, statins; mTORinhibitors, such as rapamycin or a rapamycin analog (i.e., rapalog);TGF-β signaling agents; TGF-β receptor agonists; histone deacetylaseinhibitors, such as Trichostatin A; corticosteroids; inhibitors ofmitochondrial function, such as rotenone; P38 inhibitors; NF-κβinhibitors, such as 6Bio, Dexamethasone, TCPA-1, IKK VII; adenosinereceptor agonists; prostaglandin E2 agonists (PGE2), such asMisoprostol; phosphodiesterase inhibitors, such as phosphodiesterase 4inhibitor (PDE4), such as Rolipram; proteasome inhibitors; kinaseinhibitors; G-protein coupled receptor agonists; G-protein coupledreceptor antagonists; glucocorticoids; retinoids; cytokine inhibitors;cytokine receptor inhibitors; cytokine receptor activators; peroxisomeproliferator-activated receptor antagonists; peroxisomeproliferator-activated receptor agonists; histone deacetylaseinhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3 KBinhibitors, such as TGX-221; autophagy inhibitors, such as3-Methyladenine; aryl hydrocarbon receptor inhibitors; proteasomeinhibitor I (PSI); and oxidized ATPs, such as P2X receptor blockers.Immunosuppressants also include IDO, vitamin D3, retinoic acid,cyclosporins, such as cyclosporine A, aryl hydrocarbon receptorinhibitors, resveratrol, azathiopurine (Aza), 6-mercaptopurine (6-MP),6-thioguanine (6-TG), FK506, sanglifehrin A, salmeterol, mycophenolatemofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estrioland triptolide. Other exemplary immunosuppressants include, but are notlimited, small molecule drugs, natural products, antibodies (e.g.,antibodies against CD20, CD3, CD4), biologics-based drugs,carbohydrate-based drugs, RNAi, antisense nucleic acids, aptamers,methotrexate, NSAIDs; fingolimod; natalizumab; alemtuzumab; anti-CD3;tacrolimus (FK506), abatacept, belatacept, etc. “Rapalog” refers to amolecule that is structurally related to (an analog) of rapamycin(sirolimus). Examples of rapalogs include, without limitation,temsirolimus (CCI-779), everolimus (RAD001), ridaforolimus (AP-23573),and zotarolimus (ABT-578). Additional examples of rapalogs may be found,for example, in WO Publication WO 1998/002441 and U.S. Pat. No.8,455,510, the rapalogs of which are incorporated herein by reference intheir entirety.

The immunosuppressant can be a compound that directly provides thetolerogenic effect on APCs or it can be a compound that provides thetolerogenic effect indirectly (i.e., after being processed in some wayafter administration). Immunosuppressants, therefore, include prodrugforms of any of the compounds provided herein. Furtherimmunosuppressants, are known to those of skill in the art, and theinvention is not limited in this respect. In embodiments, theimmunosuppressant may comprise any one of the agents provided herein.

“Inducing to purchase” refers to any act that suggests to an entity topurchase an antigen-presenting cell targeted immunosuppressant, a viraltransfer vector or both to achieve a beneficial effect as describedherein or to perform any one of the methods provided herein. Such actsincludes packaging an antigen-presenting cell targetedimmunosuppressant, a viral transfer vector or both that describes thebenefits of concomitant administration of an antigen-presenting celltargeted immunosuppressant and a viral transfer vector in order toattenuate an anti-viral transfer vector response, escalate transgeneexpression or allow for repeated administration of a viral transfervector. Alternatively, the packaging may describe or suggest theperformance of any one of the methods provided herein. Acts that inducean entity to purchase also include marketing an antigen-presenting celltargeted immunosuppressant, a viral transfer vector or anantigen-presenting cell targeted immunosuppressant and a viral transfervector product with information describing or suggesting the use of suchproduct for carrying out any of the beneficial effects described hereinor any one of the methods provided herein. Alternatively, the marketingincludes materials that describe or suggest the use of such product forattenuating an anti-viral transfer vector response, escalating transgeneexpression or for repeated administration of a viral transfer vector. Asa further example, acts of inducing may also comprise acts ofcommunicating information describing or suggesting any of the foregoing.The communicating is an action that can be performed in any form whetherwritten, oral, etc. If in written form, the communicating may beperformed via any medium including an electronic or a paper-basedmedium. Further, acts of inducing also include acts of distributing anantigen-presenting cell targeted immunosuppressant, a viral transfervector or both. Acts of distributing include any action to makeavailable the antigen-presenting cell targeted immunosuppressant, viraltransfer vector or both to an entity with information, packaging,marketing materials, etc. that describes, instructs or communicates anyof the benefits described herein or the steps of any one of the methodsprovided herein or the ability to attenuate an anti-viral transfervector response, escalate transgene expression or allow for repeatedadministration of a viral transfer vector. Acts of distributing includeselling, offering for sale, and transporting for sale (e.g.,transporting to pharmacies, hospitals, etc.)

“Load”, when coupled to a synthetic nanocarrier, is the amount of theimmunosuppressant coupled to the synthetic nanocarrier based on thetotal dry recipe weight of materials in an entire synthetic nanocarrier(weight/weight). Generally, such a load is calculated as an averageacross a population of synthetic nanocarriers. In one embodiment, theload on average across the synthetic nanocarriers is between 0.1% and99%. In another embodiment, the load is between 0.1% and 50%. In anotherembodiment, the load is between 0.1% and 20%. In a further embodiment,the load is between 0.1% and 10%. In still a further embodiment, theload is between 1% and 10%. In still a further embodiment, the load isbetween 7% and 20%. In yet another embodiment, the load is at least0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, atleast 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 6%, at leastat least 7%, at least 8%, at least 9%, at least 10%, at least 11%, atleast 12%, at least 13%, at least 14%, at least 15%, at least 16%, atleast 17%, at least 18%, at least 19%, at least 20%, at least 25%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% on average across the population of syntheticnanocarriers. In yet a further embodiment, the load is 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% on averageacross the population of synthetic nanocarriers. In some embodiments ofthe above embodiments, the load is no more than 25% on average across apopulation of synthetic nanocarriers. In embodiments, the load iscalculated as may be described in the Examples or as otherwise known inthe art. In some embodiments, when the form of the immunosuppressant isitself a particle or particle-like, such as a nanocrystallineimmunosuppressant, the load of immunosuppressant is the amount of theimmunosuppressant in the particles or the like (weight/weight). In suchembodiments, the load can approach 97%, 98%, 99% or more.

“Maximum dimension of a synthetic nanocarrier” means the largestdimension of a nanocarrier measured along any axis of the syntheticnanocarrier. “Minimum dimension of a synthetic nanocarrier” means thesmallest dimension of a synthetic nanocarrier measured along any axis ofthe synthetic nanocarrier. For example, for a spheroidal syntheticnanocarrier, the maximum and minimum dimension of a syntheticnanocarrier would be substantially identical, and would be the size ofits diameter. Similarly, for a cuboidal synthetic nanocarrier, theminimum dimension of a synthetic nanocarrier would be the smallest ofits height, width or length, while the maximum dimension of a syntheticnanocarrier would be the largest of its height, width or length. In anembodiment, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is equal to or greater than 100 nm. In an embodiment, a maximumdimension of at least 75%, preferably at least 80%, more preferably atleast 90%, of the synthetic nanocarriers in a sample, based on the totalnumber of synthetic nanocarriers in the sample, is equal to or less than5 μm. Preferably, a minimum dimension of at least 75%, preferably atleast 80%, more preferably at least 90%, of the synthetic nanocarriersin a sample, based on the total number of synthetic nanocarriers in thesample, is greater than 110 nm, more preferably greater than 120 nm,more preferably greater than 130 nm, and more preferably still greaterthan 150 nm. Aspects ratios of the maximum and minimum dimensions ofsynthetic nanocarriers may vary depending on the embodiment. Forinstance, aspect ratios of the maximum to minimum dimensions of thesynthetic nanocarriers may vary from 1:1 to 1,000,000:1, preferably from1:1 to 100,000:1, more preferably from 1:1 to 10,000:1, more preferablyfrom 1:1 to 1000:1, still more preferably from 1:1 to 100:1, and yetmore preferably from 1:1 to 10:1. Preferably, a maximum dimension of atleast 75%, preferably at least 80%, more preferably at least 90%, of thesynthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample is equal to or less than 3 μm, morepreferably equal to or less than 2 μm, more preferably equal to or lessthan 1 μm, more preferably equal to or less than 800 nm, more preferablyequal to or less than 600 nm, and more preferably still equal to or lessthan 500 nm. In preferred embodiments, a minimum dimension of at least75%, preferably at least 80%, more preferably at least 90%, of thesynthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample, is equal to or greater than 100nm, more preferably equal to or greater than 120 nm, more preferablyequal to or greater than 130 nm, more preferably equal to or greaterthan 140 nm, and more preferably still equal to or greater than 150 nm.Measurement of synthetic nanocarrier dimensions (e.g., effectivediameter) may be obtained, in some embodiments, by suspending thesynthetic nanocarriers in a liquid (usually aqueous) media and usingdynamic light scattering (DLS) (e.g. using a Brookhaven ZetaPALSinstrument). For example, a suspension of synthetic nanocarriers can bediluted from an aqueous buffer into purified water to achieve a finalsynthetic nanocarrier suspension concentration of approximately 0.01 to0.1 mg/mL. The diluted suspension may be prepared directly inside, ortransferred to, a suitable cuvette for DLS analysis. The cuvette maythen be placed in the DLS, allowed to equilibrate to the controlledtemperature, and then scanned for sufficient time to acquire a stableand reproducible distribution based on appropriate inputs for viscosityof the medium and refractive indicies of the sample. The effectivediameter, or mean of the distribution, is then reported. Determining theeffective sizes of high aspect ratio, or non-spheroidal, syntheticnanocarriers may require augmentative techniques, such as electronmicroscopy, to obtain more accurate measurements. “Dimension” or “size”or “diameter” of synthetic nanocarriers means the mean of a particlesize distribution, for example, obtained using dynamic light scattering.

“Measurable level” refers to any level that is above a negative controllevel or would be considered to be above background or signal noise. Ameasurable level is one that would be considered to be a levelindicating the presence of the molecule being measured.

“Non-methoxy-terminated polymer” means a polymer that has at least oneterminus that ends with a moiety other than methoxy. In someembodiments, the polymer has at least two termini that ends with amoiety other than methoxy. In other embodiments, the polymer has notermini that ends with methoxy. “Non-methoxy-terminated, pluronicpolymer” means a polymer other than a linear pluronic polymer withmethoxy at both termini. Polymeric nanoparticles as provided herein cancomprise non-methoxy-terminated polymers or non-methoxy-terminated,pluronic polymers.

“Obtaining” means an act of acquiring a material(s) by any means. Thematerial may be acquired by producing it, purchasing it, receiving it,etc. This term is intended to include “causing to obtain”. “Causing toobtain” means causing, urging, encouraging, aiding, inducing ordirecting or acting in coordination with an entity for the entity toobtain a material(s) as provided herein. In some embodiments of any oneof the methods provided herein, the method may comprise or furthercomprise any one of the steps of obtaining as described herein.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” means a pharmacologically inactive material used together witha pharmacologically active material to formulate the compositions.Pharmaceutically acceptable excipients comprise a variety of materialsknown in the art, including but not limited to saccharides (such asglucose, lactose, and the like), preservatives such as antimicrobialagents, reconstitution aids, colorants, saline (such as phosphatebuffered saline), and buffers.

“Pre-existing immunity against the viral transfer vector” refers to thepresence of antibodies, T cells and/or B cells in a subject, which cellshave been previously primed by prior exposure to antigens of the viraltransfer vector or to crossreactive antigens, including but not limitedto other viruses. In some embodiments, this pre-existing immunity is ata level that is expected to result in anti-viral transfer vector immuneresponse(s) that interferes with the efficacy of the viral transfervector. In some embodiments, this pre-existing immunity is at a levelthat is expected to result in anti-viral transfer vector immuneresponse(s) upon subsequent exposure to the viral transfer vector.Pre-existing immunity can be assessed by determining the level ofantibodies, such as neutralizing antibodies, against a viral transfervector present in a sample, such as a blood sample, from the subject.Assays for assessing the level of antibodies, such as neutralizingantibodies, are described herein at least in the Examples and are alsoknown to those of ordinary skill in the art. Such an assay is an ELISAassay. Pre-existing immunity can also be assessed by determining antigenrecall responses of immune cells, such as B or T cells, stimulated invivo or in vitro with viral transfer vector antigens presented by APCsor viral antigen epitopes presented on MHC class I or MHC class IImolecules. Assays for antigen-specific recall responses include, but arenot limited to, ELISpot, intracellular cytokine staining, cellproliferation, and cytokine production assays. Generally, these andother assays are known to those of ordinary skill in the art. In someembodiments, a subject that does not exhibit pre-existing immunityagainst the viral transfer vector is one with a level of anti-viraltransfer vector antibodies, such as neutralizing antibodies, or memory Bor T cells that would be considered to be negative. In otherembodiments, a subject that does not exhibit pre-existing immunityagainst the viral transfer vector is one with a level of an anti-viraltransfer vector response that is no more than 3 standard deviationsabove a mean negative control.

“Producing” refers to any action that results in a material being made.An act of producing includes preparing the material or processing it insome manner. In some embodiments, an act of producing includes any actthat makes that material available for use by another. This term isintended to include “causing to produce”. “Causing to produce” meanscausing, urging, encouraging, aiding, inducing or directing or acting incoordination with an entity for the entity to make a material(s) asprovided herein. In some embodiments of any one of the methods providedherein, the method may comprise or further comprise any one of the stepsof producing as described herein.

“Protocol” means a pattern of administering to a subject and includesany dosing regimen of one or more substances to a subject. Protocols aremade up of elements (or variables); thus a protocol comprises one ormore elements. Such elements of the protocol can comprise dosing amounts(doses), dosing frequency, routes of administration, dosing duration,dosing rates, interval between dosing, combinations of any of theforegoing, and the like. In some embodiments, a protocol may be used toadminister one or more compositions of the invention to one or more testsubjects. Immune responses in these test subjects can then be assessedto determine whether or not the protocol was effective in generating adesired or desired level of an immune response or therapeutic effect.Any therapeutic and/or immunologic effect may be assessed. One or moreof the elements of a protocol may have been previously demonstrated intest subjects, such as non-human subjects, and then translated intohuman protocols. For example, dosing amounts demonstrated in non-humansubjects can be scaled as an element of a human protocol usingestablished techniques such as alimetric scaling or other scalingmethods. Whether or not a protocol had a desired effect can bedetermined using any of the methods provided herein or otherwise knownin the art. For example, a sample may be obtained from a subject towhich a composition provided herein has been administered according to aspecific protocol in order to determine whether or not specific immunecells, cytokines, antibodies, etc. were reduced, generated, activated,etc. An exemplary protocol is one previously demonstrated to result in atolerogenic immune response against a viral transfer vector antigen orto achieve any one of the beneficial results described herein. Usefulmethods for detecting the presence and/or number of immune cellsinclude, but are not limited to, flow cytometric methods (e.g., FACS),ELISpot, proliferation responses, cytokine production, andimmunohistochemistry methods. Antibodies and other binding agents forspecific staining of immune cell markers, are commercially available.Such kits typically include staining reagents for antigens that allowfor FACS-based detection, separation and/or quantitation of a desiredcell population from a heterogeneous population of cells. Inembodiments, a composition as provided herein is administered to asubject using one or more or all or substantially all of the elements ofwhich a protocol is comprised, provided the selected element(s) areexpected to achieve the desired result in the subject. Such expectationmay be based on protocols determined in test subjects and scaling ifneeded. Any one of the methods provided herein may comprise or furthercomprise a step of administering a dose of the antigen-presenting celltargeted immunosuppressant alone or in combination as described hereinwith one or more doses of a viral transfer vector according to aprotocol that has been shown to attenuate an anti-viral transfer vectorimmune response or allow for the repeated administration of a viraltransfer vector. Any one of the method provided herein may comprise orfurther comprise determining such a protocol that achieves any one ofthe beneficial results described herein.

“Providing” means an action or set of actions that an individualperforms that supplies a material for practicing the invention.Providing may include acts of producing, distributing, selling, giving,making available, prescribing or administering the material. The actionor set of actions may be taken either directly oneself or indirectly.Thus, this term is intended to include “causing to provide”. “Causing toprovide” means causing, urging, encouraging, aiding, inducing ordirecting or acting in coordination with an entity for the entity tosupply a material for practicing of the present invention. In someembodiments of any one of the methods provided herein, the method maycomprise or further comprise any one of the steps of providing asdescribed herein.

“Repeat dose” or “repeat dosing” or the like means at least oneadditional dose or dosing that is administered to a subject subsequentto an earlier dose or dosing of the same material. For example, arepeated dose of a viral transfer vector is at least one additional doseof the viral transfer vector after a prior dose of the same material.While the material may be the same, the amount of the material in therepeated dose may be different from the earlier dose. For example, in anembodiment of any one of the methods or compositions provided herein,the amount of the viral transfer vector in the repeated dose may be lessthan the amount of the viral transfer vector of the earlier dose.Alternatively, in an embodiment of any one of the methods orcompositions provided herein, the repeated dose may be in an amount thatis at least equal to the amount of the viral transfer vector in theearlier dose. A repeat dose may be administered weeks, months or yearsafter the prior dose. In some embodiments of any one of the methodsprovided herein, the repeat dose or dosing is administered at least 1week after the dose or dosing that occurred just prior to the repeatdose or dosing. Repeat dosing is considered to be efficacious if itresults in a beneficial effect for the subject. Preferably, efficaciousrepeat dosing results in a beneficial effect in conjunction with anattenuated anti-viral transfer vector response.

“Selecting the doses of the viral transfer vector to be less than”refers to the selection of the doses of the viral transfer vector thatis less than the amount of the viral transfer vector that would beselected for administration to the subject if the subject were todevelop an anti-viral transfer vector immune response to the viraltransfer vector due to the repeated dosing of the viral transfer vector.This term is intended to include “causing to select”. “Causing toselect” means causing, urging, encouraging, aiding, inducing ordirecting or acting in coordination with an entity for the entity toselect the aforementioned lesser dosing. In some embodiments of any oneof the methods provided herein, the method may comprise or furthercomprise any one of the steps of selecting as described herein.

“Simultaneous” means administration at the same time or substantially atthe same time where a clinician would consider any time betweenadministrations virtually nil or negligible as to the impact on thedesired therapeutic outcome. In some embodiments, simultaneous meansthat the administrations occur with 5, 4, 3, 2, 1 or fewer minutes.

“Subject” means animals, including warm blooded mammals such as humansand primates; avians; domestic household or farm animals such as cats,dogs, sheep, goats, cattle, horses and pigs; laboratory animals such asmice, rats and guinea pigs; fish; reptiles; zoo and wild animals; andthe like. As used herein, a subject may be in one need of any one of themethods or compositions provided herein.

“Synthetic nanocarrier(s)” means a discrete object that is not found innature, and that possesses at least one dimension that is less than orequal to 5 microns in size. Albumin nanoparticles are generally includedas synthetic nanocarriers, however in certain embodiments the syntheticnanocarriers do not comprise albumin nanoparticles. In embodiments,synthetic nanocarriers do not comprise chitosan. In other embodiments,synthetic nanocarriers are not lipid-based nanoparticles. In furtherembodiments, synthetic nanocarriers do not comprise a phospholipid.

A synthetic nanocarrier can be, but is not limited to, one or aplurality of lipid-based nanoparticles (also referred to herein as lipidnanoparticles, i.e., nanoparticles where the majority of the materialthat makes up their structure are lipids), polymeric nanoparticles,metallic nanoparticles, surfactant-based emulsions, dendrimers,buckyballs, nanowires, virus-like particles (i.e., particles that areprimarily made up of viral structural proteins but that are notinfectious or have low infectivity), peptide or protein-based particles(also referred to herein as protein particles, i.e., particles where themajority of the material that makes up their structure are peptides orproteins) (such as albumin nanoparticles) and/or nanoparticles that aredeveloped using a combination of nanomaterials such as lipid-polymernanoparticles. Synthetic nanocarriers may be a variety of differentshapes, including but not limited to spheroidal, cuboidal, pyramidal,oblong, cylindrical, toroidal, and the like. Synthetic nanocarriersaccording to the invention comprise one or more surfaces. Exemplarysynthetic nanocarriers that can be adapted for use in the practice ofthe present invention comprise: (1) the biodegradable nanoparticlesdisclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymericnanoparticles of Published US Patent Application 20060002852 to Saltzmanet al., (3) the lithographically constructed nanoparticles of PublishedUS Patent Application 20090028910 to DeSimone et al., (4) the disclosureof WO 2009/051837 to von Andrian et al., (5) the nanoparticles disclosedin Published US Patent Application 2008/0145441 to Penades et al., (6)the protein nanoparticles disclosed in Published US Patent Application20090226525 to de los Rios et al., (7) the virus-like particlesdisclosed in published US Patent Application 20060222652 to Sebbel etal., (8) the nucleic acid attached virus-like particles disclosed inpublished US Patent Application 20060251677 to Bachmann et al., (9) thevirus-like particles disclosed in WO2010047839A1 or WO2009106999A2, (10)the nanoprecipitated nanoparticles disclosed in P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010), (11) apoptotic cells, apoptotic bodies or the synthetic orsemisynthetic mimics disclosed in U.S. Publication 2002/0086049, or (12)those of Look et al., Nanogel-based delivery of mycophenolic acidameliorates systemic lupus erythematosus in mice” J. ClinicalInvestigation 123(4):1741-1749 (2013).

Synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface with hydroxyl groups thatactivate complement or alternatively comprise a surface that consistsessentially of moieties that are not hydroxyl groups that activatecomplement. In a preferred embodiment, synthetic nanocarriers accordingto the invention that have a minimum dimension of equal to or less thanabout 100 nm, preferably equal to or less than 100 nm, do not comprise asurface that substantially activates complement or alternativelycomprise a surface that consists essentially of moieties that do notsubstantially activate complement. In a more preferred embodiment,synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface that activates complement oralternatively comprise a surface that consists essentially of moietiesthat do not activate complement. In embodiments, synthetic nanocarriersexclude virus-like particles. In embodiments, synthetic nanocarriers maypossess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5,1:7, or greater than 1:10.

“Therapeutic protein” means any protein that may be expressed from agene therapy transgene as provided herein. The therapeutic protein maybe one used for protein replacement or protein supplementation.Therapeutic proteins include, but are not limited to, enzymes, enzymecofactors, hormones, blood clotting factors, cytokines, growth factors,etc. Examples of other therapeutic proteins are provided elsewhereherein. A subject may be one in need of treatment with any one of thetherapeutic proteins provided herein.

“Transgene of the viral transfer vector” refers to the nucleic acidmaterial the viral transfer vector is used to transport into a cell and,once in the cell, is expressed to produce a protein or nucleic acidmolecule, respectively, such as for a therapeutic application asdescribed herein. The transgene may be a gene therapy transgene.“Expressed” or “expression” or the like refers to the synthesis of afunctional (i.e., physiologically active for the desired purpose) geneproduct after the transgene is transduced into a cell and processed bythe transduced cell. Such a gene product is also referred to herein as a“transgene expression product”. The expressed products are, therefore,the resultant protein or nucleic acid, such as an antisenseoligonucleotide or a therapeutic RNA, encoded by the transgene.

“Viral transfer vector” means a viral vector that has been adapted todeliver a transgene as provided herein. “Viral vector” refers to all ofthe viral components of a viral transfer vector that delivers atransgene. Accordingly, “viral antigen” refers to an antigen of theviral components of the viral transfer vector, such as a capsid or coatprotein, but not to the transgene or to the product it encodes. “Viraltransfer vector antigen” refers to any antigen of the viral transfervector including its viral components as well as a protein transgeneexpression product. Viral vectors are engineered to transduce one ormore desired nucleic acids into a cell. The transgene may be a genetherapy transgene. In some embodiments, the transgene is one thatencodes a protein provided herein, such as a therapeutic protein. Viralvectors can be based on, without limitation, retroviruses (e.g., murineretrovirus, avian retrovirus, Moloney murine leukemia virus (MoMuLV),Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus(MuMTV), gibbon ape leukemia virus (GaLV) and Rous Sarcoma Virus (RSV)),lentiviruses, herpes viruses, adenoviruses, adeno-associated viruses,alphaviruses, etc. Other examples are provided elsewhere herein or areknown in the art. The viral vectors may be based on natural variants,strains, or serotypes of viruses, such as any one of those providedherein. The viral vectors may also be based on viruses selected throughmolecular evolution. The viral vectors may also be engineered vectors,recombinant vectors, mutant vectors, or hybrid vectors. In someembodiments, the viral vector is a “chimeric viral vector”. In suchembodiments, this means that the viral vector is made up of viralcomponents that are derived from more than one virus or viral vector.

C. COMPOSITIONS FOR USE IN THE INVENTIVE METHODS

As mentioned above, cellular and humoral immune responses against theviral transfer vector can adversely effect the efficacy of viraltransfer vector therapeutics and can also interfere with theirreadministration, making long-term treatment impossible for manypatients. As evidenced in the art, treatment with viral transfer vectorswould not be expected to be successful for some patients due to priorexposure to a virus on which the viral transfer is based. In addition,even if a patient did not have a pre-existing immunity against a viraltransfer vector, a single administration of the viral transfer vector islikely to result in cellular and humoral immune responses, such asneutralizing antibody titers and/or the activation of memory T cells,that would not allow for successful readministration. Furthercompounding these issues is the long-term persistence of viral transfervector antigens.

Importantly, the methods and compositions provided herein have beenfound to overcome the aforementioned obstacles by attenuating immuneresponses against viral transfer vectors. The methods and compositionsprovided herein have also been found to allow for the readministrationof viral transfer vectors and provide for long-lasting tolerance againstthe viral transfer vector without the need for long-termimmunosuppression. Accordingly, the methods and compositions providedherein are useful for the treatment of subjects with a viral transfervector. Viral transfer vectors can be used to deliver transgenes for avariety of purposes, including for gene therapy, the methods andcompositions provided herein are also so applicable.

Transgenes

The transgene of the viral transfer vectors provided herein may be agene therapy transgene and encode any protein or a portion thereofbeneficial to a subject, such as one with a disease or disorder. Theprotein may be an extracellular, intracellular or membrane-boundprotein. Generally, the protein is a therapeutic protein, and thesubject to which the gene therapy transgene is administered by way of aviral transfer vector has a disease or disorder whereby the subject'sendogenous version of the protein is defective or produced in limitedamounts or not at all. Thus, the subject may be one with any one of thediseases or disorders as provided herein, and the transgene is one thatencodes any one of the therapeutic proteins or portion thereof asprovided herein.

Examples of therapeutic proteins include, but are not limited to,infusible or injectable therapeutic proteins, enzymes, enzyme cofactors,hormones, blood or blood coagulation factors, cytokines and interferons,growth factors, adipokines, etc.

Examples of infusible or injectable therapeutic proteins include, forexample, Tocilizumab (Roche/Actemra®), alpha-1 antitrypsin (Kamada/AAT),Hematide® (Affymax and Takeda, synthetic peptide), albinterferon alfa-2b(Novartis/Zalbin™), Rhucin® (Pharming Group, C1 inhibitor replacementtherapy), tesamorelin (Theratechnologies/Egrifta, synthetic growthhormone-releasing factor), ocrelizumab (Genentech, Roche and Biogen),belimumab (GlaxoSmithKline/Benlysta®), pegloticase (SavientPharmaceuticals/Krystexxa™), taliglucerase alfa (Protalix/Uplyso),agalsidase alfa (Shire/Replagal®), and velaglucerase alfa (Shire).

Examples of enzymes include lysozyme, oxidoreductases, transferases,hydrolases, lyases, isomerases, asparaginases, uricases, glycosidases,proteases, nucleases, collagenases, hyaluronidases, heparinases,heparanases, kinases, phosphatases, lysins and ligases. Other examplesof enzymes include those that used for enzyme replacement therapyincluding, but not limited to, imiglucerase (e.g., CEREZYME™),a-galactosidase A (a-gal A) (e.g., agalsidase beta, FABRYZYME™), acida-glucosidase (GAA) (e.g., alglucosidase alfa, LUMIZYME™, MYOZYME™), andarylsulfatase B (e.g., laronidase, ALDURAZYME™, idursulfase, ELAPRASE™,arylsulfatase B, NAGLAZYME™).

Examples of hormones include Melatonin (N-acetyl-5-methoxytryptamine),Serotonin, Thyroxine (or tetraiodothyronine) (a thyroid hormone),Triiodothyronine (a thyroid hormone), Epinephrine (or adrenaline),Norepinephrine (or noradrenaline), Dopamine (or prolactin inhibitinghormone), Antimullerian hormone (or mullerian inhibiting factor orhormone), Adiponectin, Adrenocorticotropic hormone (or corticotropin),Angiotensinogen and angiotensin, Antidiuretic hormone (or vasopressin,arginine vasopressin), Atrial-natriuretic peptide (or atriopeptin),Calcitonin, Cholecystokinin, Corticotropin-releasing hormone,Erythropoietin, Follicle-stimulating hormone, Gastrin, Ghrelin,Glucagon, Glucagon-like peptide (GLP-1), GIP, Gonadotropin-releasinghormone, Growth hormone-releasing hormone, Human chorionic gonadotropin,Human placental lactogen, Growth hormone, Inhibin, Insulin, Insulin-likegrowth factor (or somatomedin), Leptin, Luteinizing hormone, Melanocytestimulating hormone, Orexin, Oxytocin, Parathyroid hormone, Prolactin,Relaxin, Secretin, Somatostatin, Thrombopoietin, Thyroid-stimulatinghormone (or thyrotropin), Thyrotropin-releasing hormone, Cortisol,Aldosterone, Testosterone, Dehydroepiandrosterone, Androstenedione,Dihydrotestosterone, Estradiol, Estrone, Estriol, Progesterone,Calcitriol (1,25-dihydroxyvitamin D3), Calcidiol (25-hydroxyvitamin D3),Prostaglandins, Leukotrienes, Prostacyclin, Thromboxane, Prolactinreleasing hormone, Lipotropin, Brain natriuretic peptide, NeuropeptideY, Histamine, Endothelin, Pancreatic polypeptide, Renin, and Enkephalin.

Examples of blood or blood coagulation factors include Factor I(fibrinogen), Factor II (prothrombin), tissue factor, Factor V(proaccelerin, labile factor), Factor VII (stable factor, proconvertin),Factor VIII (antihemophilic globulin), Factor IX (Christmas factor orplasma thromboplastin component), Factor X (Stuart-Prower factor),Factor Xa, Factor XI, Factor XII (Hageman factor), Factor XIII(fibrin-stabilizing factor), von Willebrand factor, von HeldebrantFactor, prekallikrein (Fletcher factor), high-molecular weight kininogen(HMWK) (Fitzgerald factor), fibronectin, fibrin, thrombin, antithrombin,such as antithrombin III, heparin cofactor II, protein C, protein S,protein Z, protein Z-related protease inhibitot (ZPI), plasminogen,alpha 2-antiplasmin, tissue plasminogen activator (tPA), urokinase,plasminogen activator inhibitor-1 (PAI1), plasminogen activatorinhibitor-2 (PAI2), cancer procoagulant, and epoetin alfa (Epogen,Procrit).

Examples of cytokines include lymphokines, interleukins, and chemokines,type 1 cytokines, such as IFN-γ, TGF-β, and type 2 cytokines, such asIL-4, IL-10, and IL-13.

Examples of growth factors include Adrenomedullin (AM), Angiopoietin(Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs),Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF),Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cellline-derived neurotrophic factor (GDNF), Granulocyte colony-stimulatingfactor (G-CSF), Granulocyte macrophage colony-stimulating factor(GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growthfactor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growthfactor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nervegrowth factor (NGF) and other neurotrophins, Platelet-derived growthfactor (PDGF), Thrombopoietin (TPO), Transforming growth factor alpha(TGF-α), Transforming growth factor beta (TGF-β), Tumour necrosisfactor-alpha (TNF-α), Vascular endothelial growth factor (VEGF), WntSignaling Pathway, placental growth factor (PlGF), [(Foetal BovineSomatotrophin)] (FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7.

Examples of adipokines, include leptin and adiponectin.

Additional examples of therapeutic proteins include, but are not limitedto, receptors, signaling proteins, cytoskeletal proteins, scaffoldproteins, transcription factors, structural proteins, membrane proteins,cytosolic proteins, binding proteins, nuclear proteins, secretedproteins, golgi proteins, endoplasmic reticulum proteins, mitochondrialproteins, and vesicular proteins, etc.

The transgene of the gene therapy viral transfer vectors provided hereinmay encode a functional version of any protein that through some defectin the endogenous version of which in a subject (including a defect inthe expression of the endogenous version) results in a disease ordisorder in the subject. Examples of such diseases or disorders include,but are not limited to, lysosomal storage diseases/disorders, such asSantavuori-Haltia disease (Infantile Neuronal Ceroid Lipofuscinosis Type1), Jansky-Bielschowsky Disease (late infantile neuronal ceroidlipofuscinosis, Type 2), Batten disease (juvenile neuronal ceroidlipofuscinosis, Type 3), Kufs disease (neuronal ceroid lipofuscinosis,Type 4), Von Gierke disease (glycogen storage disease, Type Ia),glycogen storage disease, Type Ib, Pompe disease (glycogen storagedisease, Type II), Forbes or Cori disease (glycogen storage disease,Type III), mucolipidosis II (I-Cell disease), mucolipidosis III(Pseudo-Hurler polydystrophy), mucolipdosis IV (sialolipidosis),cystinosis (adult nonnephropathic type), cystinosis (infantilenephropathic type), cystinosis (juvenile or adolescent nephropathic),Salla disease/infantile sialic acid storage disorder, and saposindeficiencies; disorders of lipid and sphingolipid degradation, such asGM1 gangliosidosis (infantile, late infantile/juvenile, andadult/chronic), Tay-Sachs disease, Sandhoff disease, GM2 gangliodisosis,Ab variant, Fabry disease, Gaucher disease, Types I, II and III,metachromatic leukidystrophy, Krabbe disease (early and late onset),Neimann-Pick disease, Types A, B, C1, and C2, Farber disease, and Wolmandisease (cholesteryl esther storage disease); disorders ofmucopolysaccharide degradation, such as Hurler syndrome (MPSI), Scheiesyndrome (MPS IS), Hurler-Scheie syndrome (MPS IH/S), Hunter syndrome(MPS II), Sanfillippo A syndrome (MPS IIIA), Sanfillippo B syndrome (MPSIIIB), Sanfillippo C syndrome (MPS IIIC), Sanfillippo D syndrome (MPSIIID), Morquio A syndrome (MPS IVA), Morquio B syndrome (MPS IVB),Maroteaux-Lamy syndrome (MPS VI), and Sly syndrome (MPS VII); disordersof glycoprotein degradation, such as alpha mannosidosis, betamannosidosis, fucosidosis, asparylglucosaminuria, mucolipidosis I(sialidosis), galactosialidosis, Schindler disease, and Schindlerdisease, Type II/Kanzaki disease; and leukodystrophy diseases/disorders,such as abetalipoproteinemia, neonatal adrenoleukodystrophy, Canavandisease, cerebrotendinous xanthromatosis, Pelizaeus Merzbacher disease,Tangier disease, Refum disease, infantile, and Refum disease, classic.

Additional examples of such diseases/disorders of a subject as providedherein include, but are not limited to, acid maltase deficiency (e.g.,Pompe disease, glycogenosis type 2, lysosomal storage disease);carnitine deficiency; carnitine palmityl transferase deficiency;debrancher enzyme deficiency (e.g., Cori or Forbes disease, glycogenosistype 3); lactate dehydrogenase deficiency (e.g., glycogenosis type 11);myoadenylate deaminase deficiency; phosphofructokinase deficiency (e.g.,Tarui disease, glycogenosis type 7); phosphogylcerate kinase deficiency(e.g., glycogenosis type 9); phosphogylcerate mutase deficiency (e.g.,glycogenosis type 10); phosphorylase deficiency (e.g., McArdle disease,myophosphorylase deficiency, glycogenosis type 5); Gaucher's Disease(e.g., chromosome 1, enzyme glucocerebrosidase affected); Achondroplasia(e.g., chromosome 4, fibroblast growth factor receptor 3 affected);Huntington's Disease (e.g., chromosome 4, huntingtin); Hemochromatosis(e.g., chromosome 6, HFE protein); Cystic Fibrosis (e.g., chromosome 7,CFTR); Friedreich's Ataxia (chromosome 9, frataxin); Best Disease(chromosome 11, VMD2); Sickle Cell Disease (chromosome 11, hemoglobin);Phenylketoniuria (chromosome 12, phenylalanine hydroxylase); Marfan'sSyndrome (chromosome 15, fibrillin); Myotonic Dystophy (chromosome 19,dystophia myotonica protein kinase); Adrenoleukodystrophy (x-chromosome,lignoceroyl-CoA ligase in peroxisomes); Duchene's Muscular Dystrophy(x-chromosome, dystrophin); Rett Syndrome (x-chromosome,methylCpG-binding protein 2); Leber's Hereditary Optic Neuropathy(mitochondria, respiratory proteins); Mitochondria Encephalopathy,Lactic Acidosis and Stroke (MELAS) (mitochondria, transfer RNA); andEnzyme deficiencies of the Urea Cycle.

Still additional examples of such diseases or disorders include, but arenot limited to, Sickle Cell Anemia, Myotubular Myopathy, Hemophilia B,Lipoprotein lipase deficiency, Ornithine Transcarbamylase Deficiency,Crigler-Najjar Syndrome, Mucolipidosis IV, Niemann-Pick A, Sanfilippo A,Sanfilippo B, Sanfilippo C, Sanfilippo D, b-thalassaemia and DuchenneMuscular Dystrophy. Still futher examples of diseases or disordersinclude those that are the result of defects in lipid and sphingolipiddegradation, mucopolysaccharide degradation, glycoprotein degradation,leukodystrophies, etc.

The functional versions of the defective proteins of any one of thedisease or disorders provided herein may be encoded by the transgene ofa gene therapy viral transfer vector and are also considered therapeuticproteins. It follows that therapeutic proteins also includeMyophosphorylase, glucocerebrosidase, fibroblast growth factor receptor3, huntingtin, HFE protein, CFTR, frataxin, VMD2, hemoglobin,phenylalanine hydroxylase, fibrillin, dystophia myotonica proteinkinase, lignoceroyl-CoA ligase, dystrophin, methylCpG-binding protein 2,Beta hemoglobin, Myotubularin, Cathepsin A, Factor IX, Lipoproteinlipase, Beta galactosidase, Ornithine Transcarbamylase,Iduronate-2-Sulfatase, Acid-Alpha Glucosidase,UDP-glucuronosyltransferase 1-1, GlcNAc-1-phosphotransferase,GlcNAc-1-phosphotransferase, Mucolipin-1, Microsomal triglyceridetransfer protein, Sphingomyelinase, Acid ceramidase, Lysosomal acidlipase, Alpha-L-iduronidase, Heparan N-sulfatase,alpha-N-acetylglucosaminidase, acetyl-CoA alpha-glucosaminideacetyltransferase, N-acetylglucosamine 6-sulfatase,N-acetylgalactosamine-6 sulfatase, Alpha-mannosidase,Alpha-galactosidase A, Cystic fibrosis conductance transmembraneregulator, and respiratory proteins.

As further examples, therapeutic proteins also include functionalversions of proteins associated with disorders of lipid and sphingolipiddegradation (e.g., β-Galactosidase-1, β-Hexosaminidase A,β-Hexosaminidases A and B, GM2 Activator Protein, 8-Galactosidase A,Glucocerebrosidase, Glucocerebrosidase, Glucocerebrosidase,Arylsulfatase A, Galactosylceramidase, Sphingomyelinase,Sphingomyelinase, NPC1, HE1 protein (Cholesterol Trafficking Defect),Acid Ceramidase, Lysosomal Acid Lipase); disorders of mucopolysaccharidedegradation (e.g., L-Iduronidase, L-Iduronidase, L-Iduronidase,Iduronate Sulfatase, Heparan N-Sulfatase, N-Acetylglucosaminidase,Acetyl-CoA-Glucosaminidase, Acetyltransferase,Acetylglucosamine-6-Sulfatase, Galactosamine-6-Sulfatase, ArylsulfataseB, Glucuronidase); disorders of glycoprotein degradation (e.g.,Mannosidase, mannosidase, 1-fucosidase, Aspartylglycosaminidase,Neuraminidase, Lysosomal protective protein, Lysosomal8-N-acetylgalactosaminidase, Lysosomal 8-N-acetylgalactosaminidase);lysosomal storage disorders (e.g., Palmitoyl-protein thioesterase, atleast 4 subtypes, Lysosomal membrane protein, Unknown,Glucose-6-phosphatase, Glucose-6-phosphate translocase, Acid maltase,Debrancher enzyme amylo-1,6 glucosidase,N-acetylglucosamine-1-phosphotransferase,N-acetylglucosamine-1-phosphotransferase, Ganglioside sialidase(neuraminidase), Lysosomal cystine transport protein, Lysosomal cystinetransport protein, Lysosomal cystine transport protein, Sialic acidtransport protein Saposins, A, B, C, D) and leukodystrophies (e.g.,Microsomal triglyceride transfer protein/apolipoprotein B, Peroxisomalmembrane transfer protein, Peroxins, Aspartoacylase,Sterol-27-hydroxlase, Proteolipid protein, ABC1 transporter, Peroxisomemembrane protein 3 or Peroxisome biogenesis factor 1, Phytanic acidoxidase).

The sequence of a transgene may also include an expression controlsequence. Expression control DNA sequences include promoters, enhancers,and operators, and are generally selected based on the expressionsystems in which the expression construct is to be utilized. In someembodiments, promoter and enhancer sequences are selected for theability to increase gene expression, while operator sequences may beselected for the ability to regulate gene expression. The transgene mayalso include sequences that facilitate, and preferably promote,homologous recombination in a host cell. The transgene may also includesequences that are necessary for replication in a host cell.

Exemplary expression control sequences include promoter sequences, e.g.,cytomegalovirus promoter; Rous sarcoma virus promoter; and simian virus40 promoter; as well as any other types of promoters that are disclosedelsewhere herein or are otherwise known in the art. Generally, promotersare operatively linked upstream (i.e., 5′) of the sequence coding for adesired expression product. The transgene also may include a suitablepolyadenylation sequence (e.g., the SV40 or human growth hormone genepolyadenylation sequence) operably linked downstream (i.e., 3′) of thecoding sequence.

Viral Vectors

Viruses have evolved specialized mechanisms to transport their genomesinside the cells that they infect; viral vectors based on such virusescan be tailored to transduce cells to specific applications. Examples ofviral vectors that may be used as provided herein are known in the artor described herein. Suitable viral vectors include, for instance,retroviral vectors, lentiviral vectors, herpes simplex virus (HSV)-basedvectors, adenovirus-based vectors, adeno-associated virus (AAV)-basedvectors, and AAV-adenoviral chimeric vectors.

The viral transfer vectors provided herein may be based on a retrovirus.Retrovirus is a single-stranded positive sense RNA virus capable ofinfecting a wide variety of host cells. Upon infection, the retroviralgenome integrates into the genome of its host cell, using its ownreverse transcriptase enzyme to produce DNA from its RNA genome. Theviral DNA is then replicated along with host cell DNA, which translatesand transcribes the viral and host genes. A retroviral vector can bemanipulated to render the virus replication-incompetent. As such,retroviral vectors are thought to be particularly useful for stable genetransfer in vivo. Examples of retroviral vectors can be found, forexample, in U.S. Publication Nos. 20120009161, 20090118212, and20090017543, the viral vectors and methods of their making beingincorporated by reference herein in their entirety.

Lentiviral vectors are examples of retroviral vectors that can be usedfor the production of a viral transfer vector as provided herein.Lentiviruses have the ability to infect non-dividing cells, a propertythat constitute a more efficient method of a gene delivery vector (see,e.g., Durand et al., Viruses. 2011 February; 3(2): 132-159). Examples oflentiviruses include HIV (humans), simian immunodeficiency virus (SIV),feline immunodeficiency virus (FIV), equine infectious anemia virus(EIAV) and visna virus (ovine lentivirus). Unlike other retroviruses,HIV-based vectors are known to incorporate their passenger genes intonon-dividing cells. Examples of lentiviral vectors can be found, forexample, in U.S. Publication Nos. 20150224209, 20150203870, 20140335607,20140248306, 20090148936, and 20080254008, the viral vectors and methodsof their making being incorporated by reference herein in theirentirety.

Herpes simplex virus (HSV)-based viral vectors are also suitable for useas provided herein. Many replication-deficient HSV vectors contain adeletion to remove one or more intermediate-early genes to preventreplication. Advantages of the herpes vector are its ability to enter alatent stage that can result in long-term DNA expression, and its largeviral DNA genome that can accommodate exogenous DNA up to 25 kb. For adescription of HSV-based vectors, see, for example, U.S. Pat. Nos.5,837,532, 5,846,782, 5,849,572, and 5,804,413, and International PatentApplications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583, thedescription of which viral vectors and methods of their making beingincorporated by reference in its entirety.

Adenoviruses (Ads) are nonenveloped viruses that can transfer DNA invivo to a variety of different target cell types. The virus can be madereplication-deficient by deleting select genes required for viralreplication. The expendable non-replication-essential E3 region is alsofrequently deleted to allow additional room for a larger DNA insert.Viral transfer vectors can be based on adenoviruses. Adenoviral transfervectors can be produced in high titers and can efficiently transfer DNAto replicating and non-replicating cells. Unlike lentivirus, adenoviralDNA does not integrate into the genome and therefore is not replicatedduring cell division, instead they replicate in the nucleus of the hostcell using the host's replication machinery.

The adenovirus on which a viral transfer vector may be based may be fromany origin, any subgroup, any subtype, mixture of subtypes, or anyserotype. For instance, an adenovirus can be of subgroup A (e.g.,serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16,21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6),subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33,36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g.,serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49 and51), or any other adenoviral serotype. Adenoviral serotypes 1 through 51are available from the American Type Culture Collection (ATCC, Manassas,Va.). Non-group C adenoviruses, and even non-human adenoviruses, can beused to prepare replication-deficient adenoviral vectors. Non-group Cadenoviral vectors, methods of producing non-group C adenoviral vectors,and methods of using non-group C adenoviral vectors are disclosed in,for example, U.S. Pat. Nos. 5,801,030, 5,837,511, and 5,849,561, andInternational Patent Applications WO 97/12986 and WO 98/53087. Anyadenovirus, even a chimeric adenovirus, can be used as the source of theviral genome for an adenoviral vector. For example, a human adenoviruscan be used as the source of the viral genome for areplication-deficient adenoviral vector. Further examples of adenoviralvectors can be found in U.S. Publication Nos. 20150093831, 20140248305,20120283318, 20100008889, 20090175897 and 20090088398, the descriptionof which viral vectors and methods of their making being incorporated byreference in its entirety.

The viral transfer vectors provided herein can also be based onadeno-associated viruses (AAVs). AAV vectors have been of particularinterest for use in therapeutic applications such as those describedherein. AAV is a DNA virus, which is not known to cause human disease.Generally, AAV requires co-infection with a helper virus (e.g., anadenovirus or a herpes virus), or expression of helper genes, forefficient replication. AAVs have the ability to stably infect host cellgenomes at specific sites, making them more predictable thanretroviruses; however, generally, the cloning capacity of the vector is4.9 kb. AAV vectors that have been used in gene therapy applicationsgenerally have had approximately 96% of the parental genome deleted,such that only the terminal repeats (ITRs), which contain recognitionsignals for DNA replication and packaging, remain. For a description ofAAV-based vectors, see, for example, U.S. Pat. Nos. 8,679,837,8,637,255, 8,409,842, 7,803,622, and 7,790,449, and U.S. PublicationNos. 20150065562, 20140155469, 20140037585, 20130096182, 20120100606,and 20070036757, the viral vectors of which and methods or their makingbeing incorporated herein by reference in their entirety. The AAVvectors may be recombinant AAV vectors. The AAV vectors may also beself-complementary (sc) AAV vectors, which are described, for example,in U.S. Patent Publications 2007/01110724 and 2004/0029106, and U.S.Pat. Nos. 7,465,583 and 7,186,699, the vectors and methods of productionof which are herein incorporated by reference.

The adeno-associated virus on which a viral transfer vector may be ofany serotype or a mixture of serotypes. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. Forexample, when the viral transfer vector is based on a mixture ofserotypes, the viral transfer vector may contain the capsid signalsequences taken from one AAV serotype (for example selected from any oneof AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11) and packagingsequences from a different serotype (for example selected from any oneof AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11). In someembodiments of any one of the methods or compositions provided herein,therefore, the AAV vector is an AAV 2/8 vector. In other embodiments ofany one of the methods or compositions provided herein, the AAV vectoris an AAV 2/5 vector.

The viral transfer vectors provided herein may also be based on analphavirus. Alphaviruses include Sindbis (and VEEV) virus, Aura virus,Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus,Chikungunya virus, Eastern equine encephalitis virus, Everglades virus,Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus,Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus,Mucambo virus, Ndumu virus, O′nyong-nyong virus, Pixuna virus, Rio Negrovirus, Ross River virus, Salmon pancreas disease virus, Semliki Forestvirus, Southern elephant seal virus, Tonate virus, Trocara virus, Unavirus, Venezuelan equine encephalitis virus, Western equine encephalitisvirus, and Whataroa virus. Generally, the genome of such viruses encodenonstructural (e.g., replicon) and structural proteins (e.g., capsid andenvelope) that can be translated in the cytoplasm of the host cell. RossRiver virus, Sindbis virus, Semliki Forest virus (SFV), and Venezuelanequine encephalitis virus (VEEV) have all been used to develop viraltransfer vectors for transgene delivery. Pseudotyped viruses may beformed by combining alphaviral envelope glycoproteins and retroviralcapsids. Examples of alphaviral vectors can be found in U.S. PublicationNos. 20150050243, 20090305344, and 20060177819; the vectors and methodsof their making are incorporated herein by reference in their entirety.

Antigen-Presenting Cell Targeted Immunosuppressants

Antigen-presenting cell targeted immunosuppressant can include agentsthat by virtue of their form or characteristics can result in APCtolerogenic effects. Antigen-presenting cell targeted immunosuppressantalso include agents that comprise a carrier to which animmunosuppressant is conjugated.

Antigen-presenting cell targeted immunosuppressants includenegatively-charged particles, such as polystyrene, PLGA, or diamondparticles of a certain size and zeta potential, such as those describedin U.S. Publication No. 20150010631, the description of such particlesand methods of their production being incorporated herein by reference.Such particles may have any particle shape or conformation. However, insome embodiments it is preferred to use particles that are less likelyto clump in vivo. In one embodiment, these particles have a sphericalshape. Generally, it is not necessary for such particles to be uniformin size, although such particles must generally be of a size sufficientto trigger phagocytosis in an antigen-presenting cell or other MPS cell.Preferably, these particles are microscopic or nanoscopic in size, inorder to enhance solubility, avoid possible complications caused byaggregation in vivo and to facilitate pinocytosis.

These particles may an average diameter of from about 0.1 μm to about 10μm, about 0.2 μm to about 2 μm, about 0.3 μm to about 5 μm, or about 0.5μm to about 3 μm. In some embodiments, these particles may have anaverage diameter of about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4μm, about 0.5 μm, about 1.0 μm, about 1.5 μm, about 2.0 μm, about 2.5μm, about 3.0 μm, about 3.5 μm, about 4.0 μm, about 4.5 μm, or about 5.0μm. These particles need not be of uniform diameter, and apharmaceutical formulation may contain a plurality of particles with amixture of particle sizes.

In some embodiments, these particles are non-metallic. In theseembodiments, these particles may be formed from a polymer. In apreferred embodiment, these particles are biodegradable. Examples ofsuitable particles include polystyrene particles, PLGA particles,PLURONICS stabilized polypropylene sulfide particles, and diamondparticles. Additionally, these particles can be formed from a wide rangeof other materials. For example, these particles may be composed ofglass, silica, polyesters of hydroxy carboxylic acids, polyanhydrides ofdicarboxylic acids, or copolymers of hydroxy carboxylic acids anddicarboxylic acids. More generally, these particles may be composed ofother materials as described in U.S. Publication No. 20150010631.

The particles generally possess a particular zeta potential. In certainembodiments, the zeta potential is negative. The zeta potential may beless than about −100 mV or less than about −50 mV. In certainembodiments, the particles possess a zeta potential between −100 mV and0 mV, between −75 mV and 0 mV, between −60 mV and 0 mV, between −50 mVand 0 mV, between −40 mV and 0 mV, between −30 mV and 0 mV, between −20mV and +0 mV, between −10 mV and −0 mV, between −100 mV and −50 mV,between −75 mV and −50 mV, or between −50 mV and −40 mV.

In another embodiment, these particles also comprise one or moreantigens as provided herein. In some of these embodiments, the one ormore antigens are encapsulated in the particles.

Another example of an antigen-presenting cell targeted immunosuppressantis an immunosuppressants in nanocrystalline form, whereby the form ofthe immunosuppressant itself is a particle or particle-like. In theseembodiments, such forms mimic a virus or other foreign pathogen. Manydrugs have been nanosized and appropriate methods for producing suchdrug forms would be known to one of ordinary skill in the art. Drugnanocrystals, such as nanocrystalline rapamycin, are known to those ofordinary skill in the art (Katteboinaa, et al. 2009, InternationalJournal of PharmTech Resesarch; Vol. 1, No. 3; pp 682-694. As usedherein, a “drug nanocrystal” refers to a form of a drug (e.g., animmunosuppressant) that does not include a carrier or matrix material.In some embodiments, drug nanocrystals comprise 90%, 95%, 98%, or 99% ormore drug. Methods for producing drug nanocrystals include, withoutlimitation, milling, high pressure homogenization, precipitation, spraydrying, rapid expansion of supercritical solution (RESS), Nanoedge®technology (Baxter Healthcare), and Nanocrystal Technology™ (ElanCorporation). In some embodiments, a surfactant or a stabilizer may beused for steric or electrostatic stability of the drug nanocrystal. Insome embodiments, the nanocrystal or nanocrystalline form of animmunosuppressant may be used to increase the solubility, stability,and/or bioavailability of the immunosuppressant, particularlyimmunosuppressants that are insoluble or labile.

Antigen-presenting call targeted immunosuppressants also may be anapoptotic-body mimic and cause an associated antigen(s) to be tolerized.Such mimics are described in U.S. Publication No. 20120076831, whichmimics and methods of their making are incorporated herein by reference.The apoptotic-body mimics may be particles, beads, branched polymers,dendrimers, or liposomes. Preferably the mimic is particulate, andgenerally spherical, ellipsoidal, rod-shaped, globular, or polyhedral inshape. Alternatively, however, the mimic may be of an irregular orbranched shape. In preferred embodiments, the mimic is composed ofmaterial which is biodegradable. It is further preferred that the mimichave a net neutral or negative charge, in order to reduce non-specificbinding to cell surfaces which, in general, bear a net negative charge.Preferably the mimic surface is composed of a material that minimizesnon-specific or unwanted biological interactions. When a particle, themimic surface may be coated with a material to prevent or decreasenon-specific interactions. Steric stabilization by coating particleswith hydrophilic layers such as poly(ethylene glycol) (PEG) and itscopolymers such as PLURONICS (including copolymers of poly(ethyleneglycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol)) may reducethe non-specific interactions with proteins of the interstitium.

When particles, the mimics may be particles composed of glass, silica,polyesters of hydroxy carboxylic acids, polyanhydrides of dicarboxylicacids, or copolymers of hydroxy carboxylic acids and dicarboxylic acids.These mimics may be quantum dots, or composed of quantum dots, such asquantum dot polystyrene particles. These mimics may comprise materialsincluding polyglycolic acid polymers (PGA), polylactic acid polymers(PLA), polysebacic acid polymers (PSA), poly(lactic-co-glycolic) acidcopolymers (PLGA), poly(lactic-co-sebacic) acid copolymers (PLSA),poly(glycolic-co-sebacic) acid copolymers (PGSA), etc. The mimics mayalso be polystyrene beads.

These mimics may comprise one or more antigens. The mimics may becapable of being conjugated, either directly or indirectly, to one ormore antigens to which tolerance is desired. In some instances, themimic will have multiple binding sites in order to have multiple copiesof the antigen exposed and increase the likelihood of a tolerogenicresponse. The mimic may have one antigen on its surface or multipledifferent antigens on the surface. Alternatively, however, the mimic mayhave a surface to which conjugating moieties may be adsorbed withoutchemical bond formation.

In some embodiments, the mimics may also comprise an apoptotic signalingmolecule, although this is not necessarily required, such as withpolystyrene beads. Apoptotic signaling molecules include, but are notlimited to, the apoptosis signaling molecules described in U.S.Publication No. 20050113297, which apoptosis signaling molecules areherein incorporated by reference. Molecules suitable for use in theseparticles include molecules that target phagocytes, which includemacrophages, dendritic cells, monocytes and neutrophils. Such moleculesmay be thrombospondins or Annexin I.

Antigen-presenting cell targeted immunosuppressants may also beerythrocyte-binding therapeutics, such as those described in U.S.Publication No. 20120039989, which therapeutics and methods of theirmaking are incorporated herein by reference. As described, peptides thatspecifically bind to erythrocytes (also known as red blood cells) werediscovered. These peptides bind specifically to erythrocytes even in thepresence of other factors present in blood and can be used to createimmunotolerance. Accordingly, an erythrocyte-binding therapeuticcomprises one or more antigens to which tolerance is desired and anerythrocyte affinity ligand. The one or more antigens may be viraltransfer vector antigens as described herein, such as one or more viralantigens (e.g., of one or more viral capsid proteins). Also, the one ormore antigens may also be or include one or more antigens of anexpressed transgene as provided. The antigens may form a mixture towhich tolerance is desired.

Examples of peptides that specifically bind erythrocytes include ERY1,ERY19, ERY59, ERY64, ERY123, ERY141 and ERY162. In addition to peptidesthat bind erythrocytes, proteins, such as antibodies, for example singlechain antibodies, and antigen binding fragments thereof may also be usedas the affinity ligands. The affinity ligands may also includenucleotide aptamer ligands for erythrocyte surface components.Accordingly, aptamers can be made and used in place of other erythrocyteaffinity ligands. DNA and RNA aptamers may be used to providenon-covalent erythrocyte binding. Aptamers can be classified as DNAaptamers, RNA aptamers, or peptide aptamers. Additionally, the affinityligands may be a fusion of two or more affinity ligands, such aserythrocyte-binding peptides. Further, the components of theerythrocyte-binding therapeutics may be associated with a carrier suchas a polymersome, a liposome or micelle or some types of nanoparticles.In some embodiments, the components are encapsulated in such a carrier.In some embodiments, the carrier comprises an affinity ligand asdescribed herein and one or more antigens. In such an embodiment, theaffinity ligand and one or more antigens do not necessarily need to beconjugated to each other.

Antigen-presenting cell targeted immunosuppressants also include any oneof the immunosuppressants provided herein coupled to a carrier thattargets APCs. The carrier in some embodiments may be an antibody orantigen binding fragment thereof (or some other ligand) that is specificto an APC marker. Such markers include, but are not limited to, CD1a(R4, T6, HTA-1); CD1b (R1); CD1c (M241, R7); CD1d (R3); CD1e (R2); CD11b(αM Integrin chain, CR3, Mo1, C3niR, Mac-1); CD11c (αX Integrin, p150,95, AXb2); CDw117 (Lactosylceramide, LacCer); CD19 (B4); CD33 (gp67); CD35 (CR1, C3b/C4b receptor); CD 36 (GpIIIb, GPIV, PASIV); CD39(ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD45 (LCA, T200,B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD49d (VLA-4α, α4Integrin); CD49e (VLA-5α, α5 Integrin); CD58 (LFA-3); CD64 (FcγRI); CD72(Ly-19.2, Ly-32.2, Lyb-2); CD73 (Ecto-5′nucloticlase); CD74 (Ii,invariant chain); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD83 (HB15);CD85a (ILT5, LIR3, HL9); CD85d (ILT4, LIR2, MIR10); CD85j (ILT2, LIR1,MIR7); CD85k (ILT3, LIR5, HM18); CD86 (B7-2/B70); CD88 (C5aB); CD97(BL-KDD/F12); CD101 (IGSF2, P126, V7); CD116 (GM-CSFRα); CD120a (TMFRI,p55); CD120b (TNFRII, p75, TNFR p80); CD123 (IL-3Rα); CD139; CD148(HPTP-η, p260, DEP-1); CD150 (SLAM, IPO-3); CD156b (TACE, ADAM17, cSVP);CD157 (MoS, BST-1); CD167a (DDR1, trkE, cak); CD168 (RHAMM, IHABP,HMMR); CD169 (Sialoadhesin, Siglec-1); CD170 (Siglec-5); CD171 (L1CAM,NILE); CD172 (SIRP-1α, MyD-1); CD172b (SIRPβ); CD180 (RP105, Bgp95,Ly64); CD184 (CXCR4, NPY3R); CD193 (CCR3); CD196 (CCR6); CD197 (CCR7 (wsCDw197)); CDw197 (CCR7, EBI1, BLR2); CD200 (OX2); CD205 (DEC-205); CD206(MMR); CD207 (Langerin); CD208 (DC-LAMP); CD209 (DC-SIGN); CDw218a(IL18Rα); CDw218b (IL8Rβ); CD227 (MUC1, PUM, PEM, EMA); CD230 (PrionProtein (PrP)); CD252 (OX40L, TNF (ligand) superfamily, member 4); CD258(LIGHT, TNF (ligand) superfamily, member 14); CD265 (TRANCE-R, TNF-Rsuperfamily, member 11a); CD271 (NGFR, p75, TNFR superfamily, member16); CD273 (B7DC, PDL2); CD274 (B7H1, PDL1); CD275 (B7H2, ICOSL); CD276(B7H3); CD277 (BT3.1, B7 family: Butyrophilin 3); CD283 (TLR3, TOLL-likereceptor 3); CD289 (TLR9, TOLL-like receptor 9); CD295 (LEPR); CD298(ATP1B3, Na K ATPase β3 submit); CD300a (CMRF-35H); CD300c (CMRF-35A);CD301 (MGL1, CLECSF14); CD302 (DCL1); CD303 (BDCA2); CD304 (BDCA4);CD312 (EMR2); CD317 (BST2); CD319 (CRACC, SLAMF7); CD320 (8D6); and CD68(gp110, Macrosialin); class II MHC; BDCA-1; and Siglec-H. Methods forpreparing antibody-drug conjugates can be found in U.S. Publication No.20150231241, which methods are herein incorporated by reference. Othermethods are known to those in the art.

The antigen-presenting cell targeted immunosuppressant may also besynthetic nanocarriers that comprise any one of the immunosuppressantsas described herein. Such synthetic nanocarriers include those of U.S.Publication No. 20100151000, the synthetic nanocarriers of which, andmethods of their making, are incorporated herein by reference. Asdescribed, it was found that tolerogenic responses can be generated invivo by administering particles (e.g., liposomes or polymeric particles)comprising both a NF-κB inhibitor and an antigen. Accordingly, particlesthat comprise an inhibitor of the NF-κB pathway and one or more viraltransfer vector antigens can be used as antigen-presenting cell targetedimmunosuppressants as provided herein. In some embodiments, the particleis liposomal. In other embodiments, the particle comprises a carrierparticle, such as a metal particle (e.g., a tungsten, gold, platinum oriridium particle). In still other embodiments, the particle comprises apolymeric matrix or carrier, illustrative examples of which includebiocompatible polymeric particles (e.g., particles fabricated withpoly(lactide-co-glycolide)). In still other embodiments, the particlecomprises a ceramic or inorganic matrix or carrier.

The inhibitor of the NF-κB pathway can decrease the level or functionalactivity of a member of the NF-κB pathway, and can be selected from BTK,LYN, BCR Ig.alpha., BCR Ig.beta., Syk, Blnk, PLC.gamma.2, PKC.beta.,DAG, CARMA1, BCL10, MALT1, PI3K, PIPS, AKT, p38 MAPK, ERK, COT,IKK.alpha., IKK.beta., IKK.gamma., NIK, RelA/p65, P105/p50, c-Rel, RelB,p52, NIK, Leu13, CD81, CD19, CD21 and its ligands in the complement andcoagulation cascade, TRAF6, ubiquitin ligase, Tab2, TAK1, NEMO, NOD2,RIP2, Lck, fyn, Zap70, LAT, GRB2, SOS, CD3 zeta, Slp-76, GADS, ITK,PLC.gamma.1, PKC.theta., ICOS, CD28, SHP2, SAP, SLAM and 2B4. In someembodiments, the NF-κB pathway inhibitor decreases the level orfunctional activity of any one or more of RelA/p65, P105/p50, c-Rel,RelB or p52.

A wide variety of other synthetic nanocarriers can be used according tothe invention, and in some embodiments, coupled to an immunosuppressantto provide still other antigen-presenting cell targetedimmunosuppressants. In some embodiments, synthetic nanocarriers arespheres or spheroids. In some embodiments, synthetic nanocarriers areflat or plate-shaped. In some embodiments, synthetic nanocarriers arecubes or cubic. In some embodiments, synthetic nanocarriers are ovals orellipses. In some embodiments, synthetic nanocarriers are cylinders,cones, or pyramids.

In some embodiments, it is desirable to use a population of syntheticnanocarriers that is relatively uniform in terms of size or shape sothat each synthetic nanocarrier has similar properties. For example, atleast 80%, at least 90%, or at least 95% of the synthetic nanocarriersof any one of the compositions or methods provided, based on the totalnumber of synthetic nanocarriers, may have a minimum dimension ormaximum dimension that falls within 5%, 10%, or 20% of the averagediameter or average dimension of the synthetic nanocarriers.

Synthetic nanocarriers can be solid or hollow and can comprise one ormore layers. In some embodiments, each layer has a unique compositionand unique properties relative to the other layer(s). To give but oneexample, synthetic nanocarriers may have a core/shell structure, whereinthe core is one layer (e.g. a polymeric core) and the shell is a secondlayer (e.g. a lipid bilayer or monolayer). Synthetic nanocarriers maycomprise a plurality of different layers.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more lipids. In some embodiments, a synthetic nanocarrier maycomprise a liposome. In some embodiments, a synthetic nanocarrier maycomprise a lipid bilayer. In some embodiments, a synthetic nanocarriermay comprise a lipid monolayer. In some embodiments, a syntheticnanocarrier may comprise a micelle. In some embodiments, a syntheticnanocarrier may comprise a core comprising a polymeric matrix surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In someembodiments, a synthetic nanocarrier may comprise a non-polymeric core(e.g., metal particle, quantum dot, ceramic particle, bone particle,viral particle, proteins, nucleic acids, carbohydrates, etc.) surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).

In other embodiments, synthetic nanocarriers may comprise metalparticles, quantum dots, ceramic particles, etc. In some embodiments, anon-polymeric synthetic nanocarrier is an aggregate of non-polymericcomponents, such as an aggregate of metal atoms (e.g., gold atoms).

In some embodiments, synthetic nanocarriers may optionally comprise oneor more amphiphilic entities. In some embodiments, an amphiphilic entitycan promote the production of synthetic nanocarriers with increasedstability, improved uniformity, or increased viscosity. In someembodiments, amphiphilic entities can be associated with the interiorsurface of a lipid membrane (e.g., lipid bilayer, lipid monolayer,etc.). Many amphiphilic entities known in the art are suitable for usein making synthetic nanocarriers in accordance with the presentinvention. Such amphiphilic entities include, but are not limited to,phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine(DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides;sorbitan trioleate (Span® 85) glycocholate; sorbitan monolaurate (Span®20); polysorbate 20 (Tween® 20); polysorbate 60 (Tween® 60); polysorbate65 (Tween® 65); polysorbate 80 (Tween® 80); polysorbate 85 (Tween® 85);polyoxyethylene monostearate; surfactin; a poloxomer; a sorbitan fattyacid ester such as sorbitan trioleate; lecithin; lysolecithin;phosphatidylserine; phosphatidylinositol; sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)400-monostearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; ion pairing agents; and combinationsthereof. An amphiphilic entity component may be a mixture of differentamphiphilic entities. Those skilled in the art will recognize that thisis an exemplary, not comprehensive, list of substances with surfactantactivity. Any amphiphilic entity may be used in the production ofsynthetic nanocarriers to be used in accordance with the presentinvention.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more carbohydrates. Carbohydrates may be natural or synthetic. Acarbohydrate may be a derivatized natural carbohydrate. In certainembodiments, a carbohydrate comprises monosaccharide or disaccharide,including but not limited to glucose, fructose, galactose, ribose,lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose,arabinose, glucoronic acid, galactoronic acid, mannuronic acid,glucosamine, galatosamine, and neuramic acid. In certain embodiments, acarbohydrate is a polysaccharide, including but not limited to pullulan,cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose(HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran,cyclodextran, glycogen, hydroxyethylstarch, carageenan, glycon, amylose,chitosan, N,O-carboxylmethylchitosan, algin and alginic acid, starch,chitin, inulin, konjac, glucommannan, pustulan, heparin, hyaluronicacid, curdlan, and xanthan. In embodiments, the synthetic nanocarriersdo not comprise (or specifically exclude) carbohydrates, such as apolysaccharide. In certain embodiments, the carbohydrate may comprise acarbohydrate derivative such as a sugar alcohol, including but notlimited to mannitol, sorbitol, xylitol, erythritol, maltitol, andlactitol.

In some embodiments, synthetic nanocarriers can comprise one or morepolymers. In some embodiments, the synthetic nanocarriers comprise oneor more polymers that is a non-methoxy-terminated, pluronic polymer. Insome embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or99% (weight/weight) of the polymers that make up the syntheticnanocarriers are non-methoxy-terminated, pluronic polymers. In someembodiments, all of the polymers that make up the synthetic nanocarriersare non-methoxy-terminated, pluronic polymers. In some embodiments, thesynthetic nanocarriers comprise one or more polymers that is anon-methoxy-terminated polymer. In some embodiments, at least 1%, 2%,3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of thepolymers that make up the synthetic nanocarriers arenon-methoxy-terminated polymers. In some embodiments, all of thepolymers that make up the synthetic nanocarriers arenon-methoxy-terminated polymers. In some embodiments, the syntheticnanocarriers comprise one or more polymers that do not comprise pluronicpolymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, or 99% (weight/weight) of the polymers that make up thesynthetic nanocarriers do not comprise pluronic polymer. In someembodiments, all of the polymers that make up the synthetic nanocarriersdo not comprise pluronic polymer. In some embodiments, such a polymercan be surrounded by a coating layer (e.g., liposome, lipid monolayer,micelle, etc.). In some embodiments, elements of the syntheticnanocarriers can be attached to the polymer.

Immunosuppressants can be coupled to the synthetic nanocarriers by anyof a number of methods. Generally, the attaching can be a result ofbonding between the immunosuppressants and the synthetic nanocarriers.This bonding can result in the immunosuppressants being attached to thesurface of the synthetic nanocarriers and/or contained (encapsulated)within the synthetic nanocarriers. In some embodiments, however, theimmunosuppressants are encapsulated by the synthetic nanocarriers as aresult of the structure of the synthetic nanocarriers rather thanbonding to the synthetic nanocarriers. In preferable embodiments, thesynthetic nanocarrier comprises a polymer as provided herein, and theimmunosuppressants are attached to the polymer.

When attaching occurs as a result of bonding between theimmunosuppressants and synthetic nanocarriers, the attaching may occurvia a coupling moiety. A coupling moiety can be any moiety through whichan immunosuppressant is bonded to a synthetic nanocarrier. Such moietiesinclude covalent bonds, such as an amide bond or ester bond, as well asseparate molecules that bond (covalently or non-covalently) theimmunosuppressant to the synthetic nanocarrier. Such molecules includelinkers or polymers or a unit thereof. For example, the coupling moietycan comprise a charged polymer to which an immunosuppressantelectrostatically binds. As another example, the coupling moiety cancomprise a polymer or unit thereof to which it is covalently bonded.

In preferred embodiments, the synthetic nanocarriers comprise a polymeras provided herein. These synthetic nanocarriers can be completelypolymeric or they can be a mix of polymers and other materials.

In some embodiments, the polymers of a synthetic nanocarrier associateto form a polymeric matrix. In some of these embodiments, a component,such as an immunosuppressant, can be covalently associated with one ormore polymers of the polymeric matrix. In some embodiments, covalentassociation is mediated by a linker. In some embodiments, a componentcan be noncovalently associated with one or more polymers of thepolymeric matrix. For example, in some embodiments, a component can beencapsulated within, surrounded by, and/or dispersed throughout apolymeric matrix. Alternatively or additionally, a component can beassociated with one or more polymers of a polymeric matrix byhydrophobic interactions, charge interactions, van der Waals forces,etc. A wide variety of polymers and methods for forming polymericmatrices therefrom are known conventionally.

Polymers may be natural or unnatural (synthetic) polymers. Polymers maybe homopolymers or copolymers comprising two or more monomers. In termsof sequence, copolymers may be random, block, or comprise a combinationof random and block sequences. Typically, polymers in accordance withthe present invention are organic polymers.

In some embodiments, the polymer comprises a polyester, polycarbonate,polyamide, or polyether, or unit thereof. In other embodiments, thepolymer comprises poly(ethylene glycol) (PEG), polypropylene glycol,poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid),or a polycaprolactone, or unit thereof. In some embodiments, it ispreferred that the polymer is biodegradable. Therefore, in theseembodiments, it is preferred that if the polymer comprises a polyether,such as poly(ethylene glycol) or polypropylene glycol or unit thereof,the polymer comprises a block-co-polymer of a polyether and abiodegradable polymer such that the polymer is biodegradable. In otherembodiments, the polymer does not solely comprise a polyether or unitthereof, such as poly(ethylene glycol) or polypropylene glycol or unitthereof.

Other examples of polymers suitable for use in the present inventioninclude, but are not limited to polyethylenes, polycarbonates (e.g.poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic anhydride)),polypropylfumerates, polyamides (e.g. polycaprolactam), polyacetals,polyethers, polyesters (e.g., polylactide, polyglycolide,polylactide-co-glycolide, polycaprolactone, polyhydroxyacid (e.g.poly(β-hydroxyalkanoate))), poly(orthoesters), polycyanoacrylates,polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine,polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

In some embodiments, polymers in accordance with the present inventioninclude polymers which have been approved for use in humans by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. §177.2600, includingbut not limited to polyesters (e.g., polylactic acid,poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymersmay comprise anionic groups (e.g., phosphate group, sulphate group,carboxylate group); cationic groups (e.g., quaternary amine group); orpolar groups (e.g., hydroxyl group, thiol group, amine group). In someembodiments, a synthetic nanocarrier comprising a hydrophilic polymericmatrix generates a hydrophilic environment within the syntheticnanocarrier. In some embodiments, polymers can be hydrophobic. In someembodiments, a synthetic nanocarrier comprising a hydrophobic polymericmatrix generates a hydrophobic environment within the syntheticnanocarrier. Selection of the hydrophilicity or hydrophobicity of thepolymer may have an impact on the nature of materials that areincorporated within the synthetic nanocarrier.

In some embodiments, polymers may be modified with one or more moietiesand/or functional groups. A variety of moieties or functional groups canbe used in accordance with the present invention. In some embodiments,polymers may be modified with polyethylene glycol (PEG), with acarbohydrate, and/or with acyclic polyacetals derived frompolysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Certainembodiments may be made using the general teachings of U.S. Pat. No.5,543,158 to Gref et al., or WO publication WO2009/051837 by Von Andrianet al.

In some embodiments, polymers may be modified with a lipid or fatty acidgroup. In some embodiments, a fatty acid group may be one or more ofbutyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers,PLGA-PEG copolymers, and derivatives thereof. In some embodiments,polyesters include, for example, poly(caprolactone),poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA are characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid:glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention is characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers,polycyanoacrylates, and combinations comprising one or more of theforegoing polymers. The acrylic polymer may comprise fully-polymerizedcopolymers of acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids. Amine-containing polymers such as poly(lysine)(Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al.,1995, Bioconjugate Chem., 6:7), poly(ethylene imine) (PEI; Boussif etal., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92:7297), andpoly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl.Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate Chem., 7:703;and Haensler et al., 1993, Bioconjugate Chem., 4:372) arepositively-charged at physiological pH, form ion pairs with nucleicacids. In embodiments, the synthetic nanocarriers may not comprise (ormay exclude) cationic polymers.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989,Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633;and Zhou et al., 1990, Macromolecules, 23:3399). Examples of thesepolyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J.Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam etal., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem.Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al.,1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc.,121:5633).

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al.,2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc.,123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J.Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181).More generally, a variety of methods for synthesizing certain suitablepolymers are described in Concise Encyclopedia of Polymer Science andPolymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press,1980; Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al.,Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S.Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used in accordance with the presentinvention without undergoing a cross-linking step. It is further to beunderstood that the synthetic nanocarriers may comprise blockcopolymers, graft copolymers, blends, mixtures, and/or adducts of any ofthe foregoing and other polymers. Those skilled in the art willrecognize that the polymers listed herein represent an exemplary, notcomprehensive, list of polymers that can be of use in accordance withthe present invention.

In some embodiments, synthetic nanocarriers do not comprise a polymericcomponent. In some embodiments, synthetic nanocarriers may comprisemetal particles, quantum dots, ceramic particles, etc. In someembodiments, a non-polymeric synthetic nanocarrier is an aggregate ofnon-polymeric components, such as an aggregate of metal atoms (e.g.,gold atoms).

Any immunosuppressant as provided herein can be, in some embodiments,coupled to synthetic nanocarriers, antibodies or antigen-bindingfragments thereof (or other ligand that targets an APC),erythrocyte-binding peptides, etc. Immunosuppressants include, but arenot limited to, statins; mTOR inhibitors, such as rapamycin or arapamycin analog; TGF-β signaling agents; TGF-β receptor agonists;histone deacetylase (HDAC) inhibitors; corticosteroids; inhibitors ofmitochondrial function, such as rotenone; P38 inhibitors; NF-κβinhibitors; adenosine receptor agonists; prostaglandin E2 agonists;phosphodiesterase inhibitors, such as phosphodiesterase 4 inhibitor;proteasome inhibitors; kinase inhibitors; G-protein coupled receptoragonists; G-protein coupled receptor antagonists; glucocorticoids;retinoids; cytokine inhibitors; cytokine receptor inhibitors; cytokinereceptor activators; peroxisome proliferator-activated receptorantagonists; peroxisome proliferator-activated receptor agonists;histone deacetylase inhibitors; calcineurin inhibitors; phosphataseinhibitors and oxidized ATPs. Immunosuppressants also include IDO,vitamin D3, cyclosporine A, aryl hydrocarbon receptor inhibitors,resveratrol, azathiopurine, 6-mercaptopurine, aspirin, niflumic acid,estriol, tripolide, interleukins (e.g., IL-1, IL-10), cyclosporine A,siRNAs targeting cytokines or cytokine receptors and the like.

Examples of statins include atorvastatin (LIPITOR®, TORVAST®),cerivastatin, fluvastatin (LESCOL®, LESCOL® XL), lovastatin (MEVACOR®,ALTOCOR®, ALTOPREV®), mevastatin (COMPACTIN®), pitavastatin (LIVALO®,PIAVA®), rosuvastatin (PRAVACHOL®, SELEKTINE®, LIPOSTAT®), rosuvastatin(CRESTOR®), and simvastatin (ZOCOR®, LIPEX®).

Examples of mTOR inhibitors include rapamycin and analogs thereof (e.g.,CCL-779, RAD001, AP23573, C20-methallylrapamycin (C20-Marap),C16-(S)-butylsulfonamidorapamycin (C16-BSrap),C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry &Biology 2006, 13:99-107)), AZD8055, BEZ235 (NVP-BEZ235), chrysophanicacid (chrysophanol), deforolimus (MK-8669), everolimus (RAD0001),KU-0063794, PI-103, PP242, temsirolimus, and WYE-354 (available fromSelleck, Houston, Tex., USA).

Examples of TGF-β signaling agents include TGF-β ligands (e.g., activinA, GDF1, GDF11, bone morphogenic proteins, nodal, TGF-βs) and theirreceptors (e.g., ACVR1B, ACVR1C, ACVR2A, ACVR2B, BMPR2, BMPR1A, BMPR1B,TGFβRI, TGFβRII), R-SMADS/co-SMADS (e.g., SMAD1, SMAD2, SMAD3, SMAD4,SMAD5, SMAD8), and ligand inhibitors (e.g, follistatin, noggin, chordin,DAN, lefty, LTBP1, THBS1, Decorin).

Examples of inhibitors of mitochondrial function include atractyloside(dipotassium salt), bongkrekic acid (triammonium salt), carbonyl cyanidem-chlorophenylhydrazone, carboxyatractyloside (e.g., from Atractylisgummifera), CGP-37157, (−)-Deguelin (e.g., from Mundulea sericea), F16,hexokinase II VDAC binding domain peptide, oligomycin, rotenone, Ru360,SFK1, and valinomycin (e.g., from Streptomyces fulvissimus)(EMD4Biosciences, USA).

Examples of P38 inhibitors include SB-203580(4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole),SB-239063(trans-1-(4hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxy-pyrimidin-4-yl)imidazole), SB-220025(5-(2amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole)),and ARRY-797.

Examples of NF (e.g., NK-κβ) inhibitors include IFRD1,2-(1,8-naphthyridin-2-yl)-Phenol, 5-aminosalicylic acid, BAY 11-7082,BAY 11-7085, CAPE (Caffeic Acid Phenethylester), diethylmaleate, IKK-2Inhibitor IV, IMD 0354, lactacystin, MG-132 [Z-Leu-Leu-Leu-CHO], NFκBActivation Inhibitor III, NF-κB Activation Inhibitor II, JSH-23,parthenolide, Phenylarsine Oxide (PAO), PPM-18,pyrrolidinedithiocarbamic acid ammonium salt, QNZ, RO 106-9920,rocaglamide, rocaglamide AL, rocaglamide C, rocaglamide I, rocaglamideJ, rocaglaol, (R)-MG-132, sodium salicylate, triptolide (PG490), andwedelolactone.

Examples of adenosine receptor agonists include CGS-21680 and ATL-146e.

Examples of prostaglandin E2 agonists include E-Prostanoid 2 andE-Prostanoid 4.

Examples of phosphodiesterase inhibitors (non-selective and selectiveinhibitors) include caffeine, aminophylline, IBMX(3-isobutyl-1-methylxanthine), paraxanthine, pentoxifylline,theobromine, theophylline, methylated xanthines, vinpocetine, EHNA(erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximone (PERFAN™),milrinone, levosimendon, mesembrine, ibudilast, piclamilast, luteolin,drotaverine, roflumilast (DAXAS™, DALIRESP™), sildenafil (REVATION®,VIAGRA®), tadalafil (ADCIRCA®, CIALIS®), vardenafil (LEVITRA®, STAXYN®),udenafil, avanafil, icariin, 4-methylpiperazine, and pyrazolopyrimidin-7-1.

Examples of proteasome inhibitors include bortezomib, disulfiram,epigallocatechin-3-gallate, and salinosporamide A.

Examples of kinase inhibitors include bevacizumab, BIBW 2992, cetuximab(ERBITUX®), imatinib (GLEEVEC®), trastuzumab (HERCEPTIN®), gefitinib(IRESSA®), ranibizumab (LUCENTIS®), pegaptanib, sorafenib, dasatinib,sunitinib, erlotinib, nilotinib, lapatinib, panitumumab, vandetanib,E7080, pazopanib, and mubritinib.

Examples of glucocorticoids include hydrocortisone (cortisol), cortisoneacetate, prednisone, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone acetate,deoxycorticosterone acetate (DOCA), and aldosterone.

Examples of retinoids include retinol, retinal, tretinoin (retinoicacid, RETIN-A®), isotretinoin (ACCUTANE®, AMNESTEEM®, CLARAVIS®,SOTRET®), alitretinoin (PANRETIN®), etretinate (TEGISON™) and itsmetabolite acitretin (SORIATANE®), tazarotene (TAZORAC®, AVAGE®,ZORAC®), bexarotene (TARGRETIN®), and adapalene (DIFFERIN®).

Examples of cytokine inhibitors include IL1ra, IL1 receptor antagonist,IGFBP, TNF-BF, uromodulin, Alpha-2-Macroglobulin, Cyclosporin A,Pentamidine, and Pentoxifylline (PENTOPAK®, PENTOXIL®, TRENTAL®).

Examples of peroxisome proliferator-activated receptor antagonistsinclude GW9662, PPARγ antagonist III, G335, and T0070907(EMD4Biosciences, USA).

Examples of peroxisome proliferator-activated receptor agonists includepioglitazone, ciglitazone, clofibrate, GW1929, GW7647, L-165,041, LY171883, PPARγ activator, Fmoc-Leu, troglitazone, and WY-14643(EMD4Biosciences, USA).

Examples of histone deacetylase inhibitors include hydroxamic acids (orhydroxamates) such as trichostatin A, cyclic tetrapeptides (such astrapoxin B) and depsipeptides, benzamides, electrophilic ketones,aliphatic acid compounds such as phenylbutyrate and valproic acid,hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101), LAQ824,and panobinostat (LBH589), benzamides such as entinostat (MS-275),CI994, and mocetinostat (MGCD0103), nicotinamide, derivatives of NAD,dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.

Examples of calcineurin inhibitors include cyclosporine, pimecrolimus,voclosporin, and tacrolimus.

Examples of phosphatase inhibitors include BN82002 hydrochloride,CP-91149, calyculin A, cantharidic acid, cantharidin, cypermethrin,ethyl-3,4-dephostatin, fostriecin sodium salt, MAZ51,methyl-3,4-dephostatin, NSC 95397, norcantharidin, okadaic acid ammoniumsalt from prorocentrum concavum, okadaic acid, okadaic acid potassiumsalt, okadaic acid sodium salt, phenylarsine oxide, various phosphataseinhibitor cocktails, protein phosphatase 1C, protein phosphatase 2Ainhibitor protein, protein phosphatase 2A1, protein phosphatase 2A2, andsodium orthovanadate.

Compositions according to the invention can comprise pharmaceuticallyacceptable excipients, such as preservatives, buffers, saline, orphosphate buffered saline. The compositions may be made usingconventional pharmaceutical manufacturing and compounding techniques toarrive at useful dosage forms. In an embodiment, compositions aresuspended in sterile saline solution for injection together with apreservative.

D. METHODS OF USING AND MAKING THE COMPOSITIONS

Viral transfer vectors can be made with methods known to those ofordinary skill in the art or as otherwise described herein. For example,viral transfer vectors can be constructed and/or purified using themethods set forth, for example, in U.S. Pat. No. 4,797,368 and Laughlinet al., Gene, 23, 65-73 (1983).

As an example, replication-deficient adenoviral vectors can be producedin complementing cell lines that provide gene functions not present inthe replication-deficient adenoviral vectors, but required for viralpropagation, at appropriate levels in order to generate high titers ofviral transfer vector stock. The complementing cell line can complementfor a deficiency in at least one replication-essential gene functionencoded by the early regions, late regions, viral packaging regions,virus-associated RNA regions, or combinations thereof, including alladenoviral functions (e.g., to enable propagation of adenoviralamplicons). Construction of complementing cell lines involve standardmolecular biology and cell culture techniques, such as those describedby Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubelet al., Current Protocols in Molecular Biology, Greene PublishingAssociates and John Wiley & Sons, New York, N.Y. (1994).

Complementing cell lines for producing adenoviral vectors include, butare not limited to, 293 cells (described in, e.g., Graham et al., J.Gen. Virol., 36, 59-72 (1977)), PER.C6 cells (described in, e.g.,International Patent Application WO 97/00326, and U.S. Pat. Nos.5,994,128 and 6,033,908), and 293-ORF6 cells (described in, e.g.,International Patent Application WO 95/34671 and Brough et al., J.Virol., 71, 9206-9213 (1997)). In some instances, the complementing cellwill not complement for all required adenoviral gene functions. Helperviruses can be employed to provide the gene functions in trans that arenot encoded by the cellular or adenoviral genomes to enable replicationof the adenoviral vector. Adenoviral vectors can be constructed,propagated, and/or purified using the materials and methods set forth,for example, in U.S. Pat. Nos. 5,965,358, 5,994,128, 6,033,908,6,168,941, 6,329,200, 6,383,795, 6,440,728, 6,447,995, and 6,475,757,U.S. Patent Application Publication No. 2002/0034735 A1, andInternational Patent Applications WO 98/53087, WO 98/56937, WO 99/15686,WO 99/54441, WO 00/12765, WO 01/77304, and WO 02/29388, as well as theother references identified herein. Non-group C adenoviral vectors,including adenoviral serotype 35 vectors, can be produced using themethods set forth in, for example, U.S. Pat. Nos. 5,837,511 and5,849,561, and International Patent Applications WO 97/12986 and WO98/53087.

AAV vectors may be produced using recombinant methods. Typically, themethods involve culturing a host cell which contains a nucleic acidsequence encoding an AAV capsid protein or fragment thereof; afunctional rep gene; a recombinant AAV vector composed of AAV invertedterminal repeats (ITRs) and a transgene; and sufficient helper functionsto permit packaging of the recombinant AAV vector into the AAV capsidproteins. In some embodiments, the viral transfer vector may compriseinverted terminal repeats (ITR) of AAV serotypes selected from the groupconsisting of: AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10,AAV11 and variants thereof.

The components to be cultured in the host cell to package a rAAV vectorin an AAV capsid may be provided to the host cell in trans.Alternatively, any one or more of the required components (e.g.,recombinant AAV vector, rep sequences, cap sequences, and/or helperfunctions) may be provided by a stable host cell which has beenengineered to contain one or more of the required components usingmethods known to those of skill in the art. Most suitably, such a stablehost cell can contain the required component(s) under the control of aninducible promoter. However, the required component(s) may be under thecontrol of a constitutive promoter. The recombinant AAV vector, repsequences, cap sequences, and helper functions required for producingthe rAAV of the invention may be delivered to the packaging host cellusing any appropriate genetic element. The selected genetic element maybe delivered by any suitable method, including those described herein.The methods used to construct any embodiment of this invention are knownto those with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods ofgenerating rAAV virions are well known and the selection of a suitablemethod is not a limitation on the present invention. See, e.g., K.Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

In some embodiments, recombinant AAV vectors may be produced using thetriple transfection method (e.g., as described in detail in U.S. Pat.No. 6,001,650, the contents of which relating to the triple transfectionmethod are incorporated herein by reference). Typically, the recombinantAAVs are produced by transfecting a host cell with a recombinant AAVvector (comprising a transgene) to be packaged into AAV particles, anAAV helper function vector, and an accessory function vector. Generally,an AAV helper function vector encodes AAV helper function sequences (repand cap), which function in trans for productive AAV replication andencapsidation. Preferably, the AAV helper function vector supportsefficient AAV vector production without generating any detectablewild-type AAV virions (i.e., AAV virions containing functional rep andcap genes). The accessory function vector can encode nucleotidesequences for non-AAV derived viral and/or cellular functions upon whichAAV is dependent for replication. The accessory functions include thosefunctions required for AAV replication, including, without limitation,those moieties involved in activation of AAV gene transcription, stagespecific AAV mRNA splicing, AAV DNA replication, synthesis of capexpression products, and AAV capsid assembly. Viral-based accessoryfunctions can be derived from any of the known helper viruses such asadenovirus, herpesvirus (other than herpes simplex virus type-1), andvaccinia virus.

Lentiviral vectors may be produced using any of a number of methodsknown in the art. Examples of lentiviral vectors and/or methods of theirproduction can be found, for example, in U.S. Publication Nos.20150224209, 20150203870, 20140335607, 20140248306, 20090148936, and20080254008, such lentiviral vectors and methods of production areincorporated herein by reference. As an example, when the lentiviralvector is integration-incompetent, the lentiviral genome furthercomprises an origin of replication (ori), whose sequence is dependent onthe nature of cells where the lentiviral genome has to be expressed.Said origin of replication may be from eukaryotic origin, preferably ofmammalian origin, most preferably of human origin. Since the lentiviralgenome does not integrate into the cell host genome (because of thedefective integrase), the lentiviral genome can be lost in cellsundergoing frequent cell divisions; this is particularly the case inimmune cells, such as B or T cells. The presence of an origin ofreplication can be beneficial in some instances. Vector particles may beproduced after transfection of appropriate cells, such as 293 T cells,by said plasmids, or by other processes. In the cells used for theexpression of the lentiviral particles, all or some of the plasmids maybe used to stably express their coding polynucleotides, or totransiently or semi-stably express their coding polynucleotides.

Methods for producing other viral vectors as provided herein are knownin the art and may be similar to the exemplified methods above.Moreover, viral vectors are available commercially.

In embodiments, when preparing certain antigen-presenting cell targetedimmunosuppressants, methods for attaching components to, for example,erythrocyte-binding peptides, antibodies or antigen-binding fragmentsthereof (or other ligand that targets an APC), or synthetic nanocarriersmay be useful.

In certain embodiments, the attaching can be a covalent linker. Inembodiments, immunosuppressants according to the invention can becovalently attached to the external surface via a 1,2,3-triazole linkerformed by the 1,3-dipolar cycloaddition reaction of azido groups withimmunosuppressant containing an alkyne group or by the 1,3-dipolarcycloaddition reaction of alkynes with immunosuppressants containing anazido group. Such cycloaddition reactions are preferably performed inthe presence of a Cu(I) catalyst along with a suitable Cu(I)-ligand anda reducing agent to reduce Cu(II) compound to catalytic active Cu(I)compound. This Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) canalso be referred as the click reaction.

Additionally, covalent coupling may comprise a covalent linker thatcomprises an amide linker, a disulfide linker, a thioether linker, ahydrazone linker, a hydrazide linker, an imine or oxime linker, an ureaor thiourea linker, an amidine linker, an amine linker, and asulfonamide linker.

An amide linker is formed via an amide bond between an amine on onecomponent such as an immunosuppressant with the carboxylic acid group ofa second component such as the nanocarrier. The amide bond in the linkercan be made using any of the conventional amide bond forming reactionswith suitably protected amino acids and activated carboxylic acid suchN-hydroxysuccinimide-activated ester.

A disulfide linker is made via the formation of a disulfide (S—S) bondbetween two sulfur atoms of the form, for instance, of R1-S—S—R2. Adisulfide bond can be formed by thiol exchange of a component containingthiol/mercaptan group (—SH) with another activated thiol group or acomponent containing thiol/mercaptan groups with a component containingactivated thiol group.

A triazole linker, specifically a 1,2,3-triazole of the form

wherein R1 and R2 may be any chemical entities, is made by the1,3-dipolar cycloaddition reaction of an azide attached to a firstcomponent with a terminal alkyne attached to a second component such asthe immunosuppressant. The 1,3-dipolar cycloaddition reaction isperformed with or without a catalyst, preferably with Cu(I)-catalyst,which links the two components through a 1,2,3-triazole function. Thischemistry is described in detail by Sharpless et al., Angew. Chem. Int.Ed. 41(14), 2596, (2002) and Meldal, et al, Chem. Rev., 2008, 108(8),2952-3015 and is often referred to as a “click” reaction or CuAAC.

A thioether linker is made by the formation of a sulfur-carbon(thioether) bond in the form, for instance, of R1-S—R2. Thioether can bemade by either alkylation of a thiol/mercaptan (—SH) group on onecomponent with an alkylating group such as halide or epoxide on a secondcomponent. Thioether linkers can also be formed by Michael addition of athiol/mercaptan group on one component to an electron-deficient alkenegroup on a second component containing a maleimide group or vinylsulfone group as the Michael acceptor. In another way, thioether linkerscan be prepared by the radical thiol-ene reaction of a thiol/mercaptangroup on one component with an alkene group on a second component.

A hydrazone linker is made by the reaction of a hydrazide group on onecomponent with an aldehyde/ketone group on the second component.

A hydrazide linker is formed by the reaction of a hydrazine group on onecomponent with a carboxylic acid group on the second component. Suchreaction is generally performed using chemistry similar to the formationof amide bond where the carboxylic acid is activated with an activatingreagent.

An imine or oxime linker is formed by the reaction of an amine orN-alkoxyamine (or aminooxy) group on one component with an aldehyde orketone group on the second component.

An urea or thiourea linker is prepared by the reaction of an amine groupon one component with an isocyanate or thioisocyanate group on thesecond component.

An amidine linker is prepared by the reaction of an amine group on onecomponent with an imidoester group on the second component.

An amine linker is made by the alkylation reaction of an amine group onone component with an alkylating group such as halide, epoxide, orsulfonate ester group on the second component. Alternatively, an aminelinker can also be made by reductive amination of an amine group on onecomponent with an aldehyde or ketone group on the second component witha suitable reducing reagent such as sodium cyanoborohydride or sodiumtriacetoxyborohydride.

A sulfonamide linker is made by the reaction of an amine group on onecomponent with a sulfonyl halide (such as sulfonyl chloride) group onthe second component.

A sulfone linker is made by Michael addition of a nucleophile to a vinylsulfone. Either the vinyl sulfone or the nucleophile may be on thesurface of the nanocarrier or attached to a component.

The component can also be conjugated via non-covalent conjugationmethods. For example, a negative charged immunosuppressant can beconjugated to a positive charged component through electrostaticadsorption. A component containing a metal ligand can also be conjugatedto a metal complex via a metal-ligand complex.

In embodiments, the component can be attached to a polymer, for examplepolylactic acid-block-polyethylene glycol, prior to the assembly of asynthetic nanocarrier or the synthetic nanocarrier can be formed withreactive or activatable groups on its surface. In the latter case, thecomponent may be prepared with a group which is compatible with theattachment chemistry that is presented by the synthetic nanocarriers'surface. In other embodiments, a peptide component can be attached toVLPs or liposomes using a suitable linker. A linker is a compound orreagent that capable of coupling two molecules together. In anembodiment, the linker can be a homobifuntional or heterobifunctionalreagent as described in Hermanson 2008. For example, an VLP or liposomesynthetic nanocarrier containing a carboxylic group on the surface canbe treated with a homobifunctional linker, adipic dihydrazide (ADH), inthe presence of EDC to form the corresponding synthetic nanocarrier withthe ADH linker. The resulting ADH linked synthetic nanocarrier is thenconjugated with a peptide component containing an acid group via theother end of the ADH linker on nanocarrier to produce the correspondingVLP or liposome peptide conjugate.

In embodiments, a polymer containing an azide or alkyne group, terminalto the polymer chain is prepared. This polymer is then used to prepare asynthetic nanocarrier in such a manner that a plurality of the alkyne orazide groups are positioned on the surface of that nanocarrier.Alternatively, the synthetic nanocarrier can be prepared by anotherroute, and subsequently functionalized with alkyne or azide groups. Thecomponent is prepared with the presence of either an alkyne (if thepolymer contains an azide) or an azide (if the polymer contains analkyne) group. The component is then allowed to react with thenanocarrier via the 1,3-dipolar cycloaddition reaction with or without acatalyst which covalently attaches the component to the particle throughthe 1,4-disubstituted 1,2,3-triazole linker.

If the component is a small molecule it may be of advantage to attachthe component to a polymer prior to the assembly of syntheticnanocarriers. In embodiments, it may also be an advantage to prepare thesynthetic nanocarriers with surface groups that are used to attach thecomponent to the synthetic nanocarrier through the use of these surfacegroups rather than attaching the component to a polymer and then usingthis polymer conjugate in the construction of synthetic nanocarriers.

For detailed descriptions of available conjugation methods, seeHermanson G T “Bioconjugate Techniques”, 2nd Edition Published byAcademic Press, Inc., 2008. In addition to covalent attachment thecomponent can be attached by adsorption to a pre-formed syntheticnanocarrier or it can be attached by encapsulation during the formationof the synthetic nanocarrier.

Synthetic nanocarriers may be prepared using a wide variety of methodsknown in the art. For example, synthetic nanocarriers can be formed bymethods such as nanoprecipitation, flow focusing using fluidic channels,spray drying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanomaterials have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843). Additional methods havebeen described in the literature (see, e.g., Doubrow, Ed.,“Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press,Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13;Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz etal., 1988, J. Appl. Polymer Sci., 35:755; U.S. Pat. Nos. 5,578,325 and6,007,845; P. Paolicelli et al., “Surface-modified PLGA-basedNanoparticles that can Efficiently Associate and Deliver Virus-likeParticles” Nanomedicine. 5(6):843-853 (2010)).

Materials may be encapsulated into synthetic nanocarriers as desirableusing a variety of methods including but not limited to C. Astete etal., “Synthesis and characterization of PLGA nanoparticles” J. Biomater.Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K. Avgoustakis“Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide) Nanoparticles:Preparation, Properties and Possible Applications in Drug Delivery”Current Drug Delivery 1:321-333 (2004); C. Reis et al.,“Nanoencapsulation I. Methods for preparation of drug-loaded polymericnanoparticles” Nanomedicine 2:8-21 (2006); P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010). Other methods suitable for encapsulating materials intosynthetic nanocarriers may be used, including without limitation methodsdisclosed in U.S. Pat. No. 6,632,671 to Unger issued Oct. 14, 2003.

In certain embodiments, synthetic nanocarriers are prepared by ananoprecipitation process or spray drying. Conditions used in preparingsynthetic nanocarriers may be altered to yield particles of a desiredsize or property (e.g., hydrophobicity, hydrophilicity, externalmorphology, “stickiness,” shape, etc.). The method of preparing thesynthetic nanocarriers and the conditions (e.g., solvent, temperature,concentration, air flow rate, etc.) used may depend on the materials tobe attached to the synthetic nanocarriers and/or the composition of thepolymer matrix.

If synthetic nanocarriers prepared by any of the above methods have asize range outside of the desired range, synthetic nanocarriers can besized, for example, using a sieve.

Elements of the synthetic nanocarriers may be attached to the overallsynthetic nanocarrier, e.g., by one or more covalent bonds, or may beattached by means of one or more linkers. Additional methods offunctionalizing synthetic nanocarriers may be adapted from Published USPatent Application 2006/0002852 to Saltzman et al., Published US PatentApplication 2009/0028910 to DeSimone et al., or Published InternationalPatent Application WO/2008/127532 A1 to Murthy et al.

Alternatively or additionally, synthetic nanocarriers can be attached tocomponents directly or indirectly via non-covalent interactions. Innon-covalent embodiments, the non-covalent attaching is mediated bynon-covalent interactions including but not limited to chargeinteractions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. Suchattachments may be arranged to be on an external surface or an internalsurface of a synthetic nanocarrier. In embodiments, encapsulation and/orabsorption is a form of attaching.

Compositions provided herein may comprise inorganic or organic buffers(e.g., sodium or potassium salts of phosphate, carbonate, acetate, orcitrate) and pH adjustment agents (e.g., hydrochloric acid, sodium orpotassium hydroxide, salts of citrate or acetate, amino acids and theirsalts) antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants(e.g., polysorbate 20, polysorbate 80, polyoxyethylene9-10 nonyl phenol,sodium desoxycholate), solution and/or cryo/lyo stabilizers (e.g.,sucrose, lactose, mannitol, trehalose), osmotic adjustment agents (e.g.,salts or sugars), antibacterial agents (e.g., benzoic acid, phenol,gentamicin), antifoaming agents (e.g., polydimethylsilozone),preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymericstabilizers and viscosity-adjustment agents (e.g., polyvinylpyrrolidone,poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol,polyethylene glycol, ethanol).

Compositions according to the invention may comprise pharmaceuticallyacceptable excipients. The compositions may be made using conventionalpharmaceutical manufacturing and compounding techniques to arrive atuseful dosage forms. Techniques suitable for use in practicing thepresent invention may be found in Handbook of Industrial Mixing: Scienceand Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, andSuzanne M. Kresta, 2004 John Wiley & Sons, Inc.; and Pharmaceutics: TheScience of Dosage Form Design, 2nd Ed. Edited by M. E. Auten, 2001,Churchill Livingstone. In an embodiment, compositions are suspended insterile saline solution for injection with a preservative.

It is to be understood that the compositions of the invention can bemade in any suitable manner, and the invention is in no way limited tocompositions that can be produced using the methods described herein.Selection of an appropriate method of manufacture may require attentionto the properties of the particular moieties being associated.

In some embodiments, compositions are manufactured under sterileconditions or are terminally sterilized. This can ensure that resultingcompositions are sterile and non-infectious, thus improving safety whencompared to non-sterile compositions. This provides a valuable safetymeasure, especially when subjects receiving the compositions have immunedefects, are suffering from infection, and/or are susceptible toinfection.

Administration according to the present invention may be by a variety ofroutes, including but not limited to subcutaneous, intravenous,intramuscular and intraperitoneal routes. The compositions referred toherein may be manufactured and prepared for administration, in someembodiments concomitant administration, using conventional methods.

The compositions of the invention can be administered in effectiveamounts, such as the effective amounts described elsewhere herein. Insome embodiments, the antigen-presenting cell targetedimmunosuppressants and/or viral transfer vectors are present in dosageforms in an amount effective to attenuate an anti-viral transfer vectorimmune response or allow for readministration of a viral transfer vectorto a subject. In some embodiments, the antigen-presenting cell targetedimmunosuppressants and/or viral transfer vectors are present in dosageforms in an amount effective to escalate transgene expression in asubject. In preferable embodiments, the antigen-presenting cell targetedimmunosuppressants and/or viral transfer vectors are present in dosageforms in an amount effective to reduce immune responses to the viraltransfer vector, such as when concomitantly administered to a subject.Dosage forms may be administered at a variety of frequencies. In someembodiments, repeated administration of antigen-presenting cell targetedimmunosuppressant with a viral transfer vector is undertaken.

Aspects of the invention relate to determining a protocol for themethods of administration as provided herein. A protocol can bedetermined by varying at least the frequency, dosage amount of the viraltransfer vector and antigen-presenting cell targeted immunosuppressantand subsequently assessing a desired or undesired immune response. Apreferred protocol for practice of the invention reduces an immuneresponse against the viral transfer vector, attenuates an anti-viraltransfer vector response and/or escalates transgene expression. Theprotocol comprises at least the frequency of the administration anddoses of the viral transfer vector and antigen-presenting cell targetedimmunosuppressant.

EXAMPLES Example 1 Polymeric Nanocarrier Containing Polymer-RapamycinConjugate (Prophetic)

Preparation of PLGA-Rapamycin Conjugate:

PLGA polymer with acid end group (7525 DLG1A, acid number 0.46 mmol/g,Lakeshore Biomaterials; 5 g, 2.3 mmol, 1.0 eq) is dissolved in 30 mL ofdichloromethane (DCM). N,N-Dicyclohexylcarbodimide (1.2 eq, 2.8 mmol,0.57 g) is added followed by rapamycin (1.0 eq, 2.3 mmol, 2.1 g) and4-dimethylaminopyridine (DMAP) (2.0 eq, 4.6 mmol, 0.56 g). The mixtureis stirred at rt for 2 days. The mixture is then filtered to removeinsoluble dicyclohexylurea. The filtrate is concentrated to ca. 10 mL involume and added to 100 mL of isopropyl alcohol (IPA) to precipitate outthe PLGA-rapamycin conjugate. The IPA layer is removed and the polymeris then washed with 50 mL of IPA and 50 mL of methyl t-butyl ether(MTBE). The polymer is then dried under vacuum at 35 C for 2 days togive PLGA-rapamycin as a white solid (ca. 6.5 g).

Nanocarrier containing PLGA-rapamycin is prepared as follows:

Solutions for nanocarrier formation are prepared as follows:

Solution 1: PLGA-rapamycin @ 100 mg/mL in methylene chloride. Thesolution is prepared by dissolving PLGA-rapamycin in pure methylenechloride. Solution 2: PLA-PEG @ 100 mg/mL in methylene chloride. Thesolution is prepared by dissolving PLA-PEG in pure methylene chloride.Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

A primary water-in-oil emulsion is prepared first. W1/O1 is prepared bycombining solution 1 (0.75 mL), and solution 2 (0.25 mL) in a smallpressure tube and sonicating at 50% amplitude for 40 seconds using aBranson Digital Sonifier 250. A secondary emulsion (W1/O1/W2) is thenprepared by combining solution 3 (3.0 mL) with the primary W1/O1emulsion, vortexing for 10 s, and sonicating at 30% amplitude for 60seconds using the Branson Digital Sonifier 250. The W1/O1/W2 emulsion isadded to a beaker containing 70 mM pH 8 phosphate buffer solution (30mL) and stirred at room temperature for 2 hours to allow the methylenechloride to evaporate and for the nanocarriers to form. A portion of thenanocarriers is washed by transferring the nanocarrier suspension to acentrifuge tube and centrifuging at 75,600×g and 4° C. for 35 min,removing the supernatant, and re-suspending the pellet in phosphatebuffered saline. The washing procedure is repeated, and the pellet isre-suspended in phosphate buffered saline for a final nanocarrierdispersion of about 10 mg/mL.

Example 2 Preparation of Gold Nanocarriers (AuNCs) Containing Rapamycin(Prophetic)

Preparation of HS-PEG-Rapamycin:

A solution of PEG acid disulfide (1.0 eq), rapamycin (2.0-2.5 eq), DCC(2.5 eq) and DMAP (3.0 eq) in dry DMF is stirred at rt overnight. Theinsoluble dicyclohexylurea is removed by filtration and the filtrate isadded to isopropyl alcohol (IPA) to precipitate out thePEG-disulfide-di-rapamycin ester and washed with IPA and dried. Thepolymer is then treated with tris(2-carboxyethyl)phosphine hydrochloridein DMF to reduce the PEG disulfide to thiol PEG rapamycin ester(HS-PEG-rapamycin). The resulting polymer is recovered by precipitationfrom IPA and dried as previously described and analyzed by H NMR andGPC.

Formation of Gold NCs (AuNCs):

An aq. solution of 500 mL of 1 mM HAuC14 is heated to reflux for 10 minwith vigorous stirring in a 1 L round-bottom flask equipped with acondenser. A solution of 50 mL of 40 mM of trisodium citrate is thenrapidly added to the stirring solution. The resulting deep wine redsolution is kept at reflux for 25-30 min and the heat is withdrawn andthe solution is cooled to room temperature. The solution is thenfiltered through a 0.8 μm membrane filter to give the AuNCs solution.The AuNCs are characterized using visible spectroscopy and transmissionelectron microscopy. The AuNCs are ca. 20 nm diameter capped by citratewith peak absorption at 520 nm.

AuNCs Conjugate with HS-PEG-Rapamycin:

A solution of 150 μl of HS-PEG-rapamycin (10 μM in 10 mM pH 9.0carbonate buffer) is added to 1 mL of 20 nm diameter citrate-capped goldnanocarriers (1.16 nM) to produce a molar ratio of thiol to gold of2500:1. The mixture is stirred at room temperature under argon for 1hour to allow complete exchange of thiol with citrate on the goldnanocarriers. The AuNCs with PEG-rapamycin on the surface is thenpurified by centrifuge at 12,000 g for 30 minutes. The supernatant isdecanted and the pellet containing AuNC-S-PEG-rapamycin is then pelletwashed with 1×PBS buffer. The purified Gold-PEG-rapamycin nanocarriersare then resuspend in suitable buffer for further analysis andbioassays.

Example 3 Mesoporous Silica Nanoparticles with Attached Ibuprofen(Prophetic)

Mesoporous SiO2 nanoparticle cores are created through a sol-gelprocess. Hexadecyltrimethyl-ammonium bromide (CTAB) (0.5 g) is dissolvedin deionized water (500 mL), and then 2 M aqueous NaOH solution (3.5 mL)is added to the CTAB solution. The solution is stirred for 30 min, andthen Tetraethoxysilane (TEOS) (2.5 mL) is added to the solution. Theresulting gel is stirred for 3 h at a temperature of 80° C. The whiteprecipitate which forms is captured by filtration, followed by washingwith deionized water and drying at room temperature. The remainingsurfactant is then extracted from the particles by suspension in anethanolic solution of HCl overnight. The particles are washed withethanol, centrifuged, and redispersed under ultrasonication. This washprocedure is repeated two additional times.

The SiO2 nanoparticles are then functionalized with amino groups using(3-aminopropyl)-triethoxysilane (APTMS). To do this, the particles aresuspended in ethanol (30 mL), and APTMS (50 μL) is added to thesuspension. The suspension is allowed to stand at room temperature for 2h and then is boiled for 4 h, keeping the volume constant byperiodically adding ethanol. Remaining reactants are removed by fivecycles of washing by centrifugation and redispersing in pure ethanol.

In a separate reaction, 1-4 nm diameter gold seeds are created. Allwater used in this reaction is first deionized and then distilled fromglass. Water (45.5 mL) is added to a 100 mL round-bottom flask. Whilestirring, 0.2 M aqueous NaOH (1.5 mL) is added, followed by a 1% aqueoussolution of tetrakis(hydroxymethyl)phosphonium chloride (THPC) (1.0 mL).Two minutes after the addition of THPC solution, a 10 mg/mL aqueoussolution of chloroauric acid (2 mL), which has been aged at least 15min, is added. The gold seeds are purified through dialysis againstwater.

To form the core-shell nanocarriers, the amino-functionalized SiO2nanoparticles formed above are first mixed with the gold seeds for 2 hat room temperature. The gold-decorated SiO2 particles are collectedthrough centrifugation and mixed with an aqueous solution of chloroauricacid and potassium bicarbonate to form the gold shell. The particles arethen washed by centrifugation and redispersed in water. Ibuprofen isloaded by suspending the particles in a solution of sodium ibuprofen (1mg/L) for 72 h. Free ibuprofen is then washed from the particles bycentrifugation and redispersing in water.

Example 4 Liposomes Containing Cyclosporine A (Prophetic)

The liposomes are formed using thin film hydration.1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 μmol),cholesterol (32 μmol), and cyclosporin A (6.4 μmol) are dissolved inpure chloroform (3 mL). This lipid solution is added to a 50 mLround-bottom flask, and the solvent is evaporated on a rotary evaporatorat a temperature of 60° C. The flask is then flushed with nitrogen gasto remove remaining solvent. Phosphate buffered saline (2 mL) and fiveglass beads are added to the flask, and the lipid film is hydrated byshaking at 60° C. for 1 h to form a suspension. The suspension istransferred to a small pressure tube and sonicated at 60° C. for fourcycles of 30 s pulses with a 30 s delay between each pulse. Thesuspension is then left undisturbed at room temperature for 2 h to allowfor complete hydration. The liposomes are washed by centrifugationfollowed by resuspension in fresh phosphate buffered saline.

Example 5 Synthetic Nanocarriers Comprising Rapamycin Materials

Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham,Mass. 01702; Product Catalogue # R1017). PLGA with 76% lactide and 24%glycolide content and an inherent viscosity of 0.69 dL/g was purchasedfrom SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala.35211. Product Code 7525 DLG 7A.) PLA-PEG block co-polymer with a PEGblock of approximately 5,000 Da and PLA block of approximately 40,000 Dawas purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,Birmingham, Ala. 35211; Product Code 100 DL mPEG 5000 5CE). Polyvinylalcohol (85-89% hydrolyzed) was purchased from EMD Chemicals (ProductNumber 1.41350.1001).

Method

Solutions were prepared as follows:

Solution 1: PLGA at 75 mg/mL and PLA-PEG at 25 mg/mL in methylenechloride. The solution was prepared by dissolving PLGA and PLA-PEG inpure methylene chloride.

Solution 2: Rapamycin at 100 mg/mL in methylene chloride. The solutionwas prepared by dissolving rapamycin in pure methylene chloride.

Solution 3: Polyvinyl alcohol at 50 mg/mL in 100 mM pH 8 phosphatebuffer.

An oil-in-water emulsion was used to prepare the nanocarriers. The O/Wemulsion was prepared by combining solution 1 (1 mL), solution 2 (0.1mL), and solution 3 (3 mL) in a small pressure tube and sonicating at30% amplitude for 60 seconds using a Branson Digital Sonifier 250. TheO/W emulsion was added to a beaker containing 70 mM pH 8 phosphatebuffer solution (30 mL) and stirred at room temperature for 2 hours toallow the methylene chloride to evaporate and for the nanocarriers toform. A portion of the nanocarriers was washed by transferring thenanocarrier suspension to a centrifuge tube and centrifuging at 75,000×gand 4° C. for 35 min, removing the supernatant, and re-suspending thepellet in phosphate buffered saline. The washing procedure was repeated,and the pellet was re-suspended in phosphate buffered saline for a finalnanocarrier dispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountrapamycin in the nanocarrier was determined by HPLC analysis. The totaldry-nanocarrier mass per mL of suspension was determined by agravimetric method.

Effective Diameter Rapamycin Content (nm) (% w/w) 227 6.4

Example 6 Synthetic Nanocarriers Comprising GSK1059615 Materials

GSK1059615 was purchased from MedChem Express (11 Deer Park Drive, Suite102D Monmouth Junction, N.J. 08852), product code HY-12036. PLGA with alactide:glycolide ratio of 1:1 and an inherent viscosity of 0.24 dL/gwas purchased from Lakeshore Biomaterials (756 Tom Martin Drive,Birmingham, Ala. 35211), product code 5050 DLG 2.5A. PLA-PEG-OMe blockco-polymer with a methyl ether terminated PEG block of approximately5,000 Da and an overall inherent viscosity of 0.26 DL/g was purchasedfrom Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, Ala.35211; Product Code 100 DL mPEG 5000 5K-E). Cellgro phosphate bufferedsaline 1× pH 7.4 (PBS 1×) was purchased from Corning (9345 DiscoveryBlvd. Manassas, Va. 20109), product code 21-040-CV.

Method

Solutions were prepared as follows:

Solution 1: PLGA (125 mg), and PLA-PEG-OMe (125 mg), were dissolved in10 mL of acetone. Solution 2: GSK1059615 was prepared at 10 mg in 1 mLof N-methyl-2-pyrrolidinone (NMP).

Nanocarriers were prepared by combining Solution 1 (4 mL) and Solution 2(0.25 mL) in a small glass pressure tube and adding the mixture dropwise to a 250 mL round bottom flask containing 20 mL of ultra-pure waterunder stirring. The flask was mounted onto a rotary evaporation device,and the acetone was removed under reduced pressure. A portion of thenanocarriers was washed by transferring the nanocarrier suspension tocentrifuge tubes and centrifuging at 75,600 rcf and 4° C. for 50minutes, removing the supernatant, and re-suspending the pellet in PBS1×. The washing procedure was repeated, and the pellet was re-suspendedin PBS 1× to achieve a nanocarrier suspension having a nominalconcentration of 10 mg/mL on a polymer basis. The washed nanocarriersolution was then filtered using 1.2 μm PES membrane syringe filtersfrom Pall, part number 4656. An identical nanocarrier solution wasprepared as above, and pooled with the first after the filtration step.The homogenous suspension was stored frozen at −20° C.

Nanocarrier size was determined by dynamic light scattering. The amountof GSK1059615 in the nanocarrier was determined by UV absorption at 351nm. The total dry-nanocarrier mass per mL of suspension was determinedby a gravimetric method.

Effective Diameter GSK1059615 Content (nm) (% w/w) 143 1.02

Example 7 Erythrocyte-Binding Therapeutic with a Viral Transfer VectorAntigen (Prophetic)

An erythrocyte-binding therapeutic is prepared based on the teachings ofU.S. Publication No. 20120039989 and used as an antigen-presenting celltargeted immunosuppressant. The erythrocyte-binding therapeutic maycomprise any one of ERY1, ERY19, ERY59, ERY64, ERY123, ERY141 and ERY162and any one of the viral transfer vector antigens described herein, suchas a viral vector antigen, e.g., a capsid protein (or peptide antigenderived therefrom), or a protein (or peptide antigen derived therefrom),such as a therapeutic protein (or peptide antigen derived therefrom)that is encoded by a transgene as described herein.

Example 8 Particles Containing an Inhibitor of the NF-kB Pathway(Prophetic)

An antigen-presenting cell targeted immunosuppressant is preparedaccording to the teachings of U.S. Publication No. 20100151000. Theparticle may be a liposome or polymeric particle and comprises any oneof the immunosuppressants provided herein or any one of the inhibitorsof the NF-kB pathway provided in U.S. Publication No. 20100151000, whichinhibitors are incorporated herein by reference in their entirety. Inaddition, the liposome or polymeric particle may further comprise anyone of the viral transfer vector antigens described herein, such as aviral vector antigen, e.g., a capsid protein (or peptide antigen derivedtherefrom), or a protein (or peptide antigen derived therefrom), such asa therapeutic protein (or peptide antigen derived therefrom), that isencoded by a transgene as described herein.

Example 9 Adenoviral Transfer Vector with a Gene Therapy Transgene(Prophetic)

An adenoviral transfer vector is generated according to the methodsprovided in U.S. Patent Publication 2004/0005293. Such a vector maycomprise any one of the transgenes as provided herein. For example, anAd-AAT-hFVIII vector that expresses the human B-domain deleted FVIIIcDNA from the human alfa1-antitrypsin promoter (AAT) is prepared. AnHPRT stuffer fragment is employed to optimize vector size and to avoidvector rearrangements (Parks R J, Graham F L. A helper-dependent systemfor adenovirus vector production helps define a lower limit forefficient DNA packaging. J Virol. 1997, 71:3293-3298). The Cre66packaging cell line is used.

Example 10 Concomitant Administration of a Viral Transfer Vector with aGene Therapy Transgene with Synthetic Nanocarriers Coupled toImmunosuppressant (Prophetic)

The viral transfer vector of any one of the Examples, such as Example 9,is administered concomitantly, such as on the same day, as any one ofthe antigen-presenting cell targeted immunosuppressants provided herein,such as in Examples 1-8 or 12, to subjects recruited for a clinicaltrial. One or more immune responses against the viral transfer vector isevaluated. The level(s) of the one or more immune responses against theviral transfer vector can be evaluated by comparison with the level(s)of the one or more immune responses in the subjects, or another group ofsubjects, administered the viral transfer vector in the absence of theantigen-presenting cell targeted immunosuppressant, such as whenadministered the viral transfer vector alone. In embodiments, repeatedconcomitant administration is evaluated in a similar manner.

In an application of the information established during such trials, theviral transfer vector and antigen-presenting cell targetedimmunosuppressant can be administered concomitantly to subjects in needof viral transfer vectors when such subjects are expected to have anundesired immune response against the viral transfer vector when notadministered concomitantly with the antigen-presenting cell targetedimmunosuppressant. In a further embodiment, a protocol using theinformation established during the trials can be prepared to guide theconcomitant dosing of the viral transfer vector and syntheticnanocarriers of subjects in need of treatment with a viral transfervector and have or are expected to have an undesired immune responseagainst the viral transfer vector without the benefit of theantigen-presenting cell targeted immunosuppressant. The protocol soprepared can then be used to treat subjects, particularly humansubjects.

Example 11 Administration of a Viral Transfer Vector with a Gene TherapyTransgene with Synthetic Nanocarriers Coupled to Immunosuppressant

Two successive intravenous (i.v.) inoculations of adeno-associated virusexpressing recombinant green fluorescent protein (AAV-GFP) led to higherGFP expression in liver cells in vivo if nanocarrier-encapsulatedimmunosuppressant (NCs) was co-injected at boost stage.

Experimental Methods

Male C57BL/6 mice were used (5 mice/group). Animals were injected with200 μL of AAV-GFP or AAV-GFP+synthetic nanocarriers comprising rapamycin(NCs) mixture once or twice over a 21d interval at different iterations(see Table 1 below). At d33 after the first injection (=d12 after thesecond injection for those groups that were injected twice) animals weresacrificed, their livers treated with collagenase 4 (Worthington,Lakewood, N.J.), meshed and total cell suspensions analyzed by FACS forGFP expression. Briefly, tissue was initially perfused with collagenase(100 U), incubated at 37° C. (30 min), collagenase supernatant removed,and quenched with 2% FBS. Tissue samples were then cut into ˜2 mmsquares, digested (collagenase, 400 U) with repeated agitation, filtered(nylon mesh), spun down (1,500 rpm), and pellets re-suspended inice-cold 2% FBS.

At day 14 after the first injections all animals were bled and theirserum analyzed for antibodies against AAV with ELISA as follows. 96-wellplates were coated with 50 μL of AAV at 2×10⁹ vg/mL in carbonate bufferfor 92 hours, and then blocked for 2 hours with 300 μL of casein.Samples were added at a 1:40 dilution in 50 μL of casein, and incubatedfor 2 hours at RT. Rabbit Anti-mouse IgG (Jackson ImmunoResearch, WestGrove, Pa., 315-035-008) was used as a secondary antibody (0.5 μg/mL, 1hour) and then TMB substrate was added (10 min) followed by the stopsolution. Plates were then read at wavelength of 450 nm with asubtraction of background at 570 nm. Mouse monoclonal anti-AAV8 antibody(Fitzgerald, Acton, Mass., 10R-2136) served as a positive control.

Amounts of AAV-GFP:

1×10¹⁰ viral genomes (VG) at d0 prime, 5×10¹⁰ VG at d21 boost.

Amounts of Nanocarrier-Encapsulated Immunosuppressant (Rapamycin orRapa) Used:

50 μg of nanocarrier-entrapped Rapa at either prime (gr. 2, 3 and 5) orboost (gr. 3 and 4).

TABLE 1 Experimental Groups Gr. # Immunization, i.v. NCs (i.v., day 0)Boost, d. 21 1 AAV-GFP None AAV-GFP; 5 × 10¹⁰ VG (1 × 10¹⁰ VG) 2 Same 50μg of Rapa Same 3 Same Same AAV-GFP, 5 × 10¹⁰ VG + NCs 4 Same NoneAAV-GFP, 5 × 10¹⁰ VG + NCs 5 Same 50 μg of Rapa None 6 Same None None

Results

Statistically higher levels of GFP expression in the liver ofAAV-injected mice were seen if NCs was utilized at the boost stage afterprime with AAV-GFP only compared to both prime and boost with AAV-GFPonly (FIG. 1). There was also a trend towards higher GFP expression ifAAV-GFP was co-injected with NCs at both prime and boost, but due to asingle outlier it did not manifest a clear statistical superiority toprime-boost with AAV-GFP only. Utilization of NCs only at primeinjection did not result in any elevation of GFP expression (FIG. 1, gr.1 vs. gr. 2 and gr. 5 vs. gr. 6). Collectively, it appeared thatco-administration of AAV-GFP and NCs at boost drives the higher GFPexpression in animals, which received two injections of recombinant AAVaccording to the current regimen. This was pronounced if all the animalsboosted with AAV-GFP only (whether or not treated with NCs at prime) areplotted against all the animals boosted with AAV-GFP+NCs (FIG. 2) with9/10 animals boosted in presence of NCs exhibiting higher GFP expressionthan all (10/10) animals boosted without NCs (average expressionincrease in the former being >50%). Similarly, if only highly-GFPpositive liver cells were considered, utilization of NCs during boostresulted in statistically higher numbers than boost with AAV-GFP withoutNCs, whether or not NCs was utilized at the prime stage (FIG. 3). It wasalso apparent that AAV-GFP boost without NCs led to decreased GFPexpression even compared to a single prime immunization.

Separately, mice were bled at d14 and their serum tested for thepresence of antibodies to AAV. At this point, all mice had been injectedwith AAV-GFP once with or without co-administration of NCs (resulting intwo groups of 15 mice each). As seen in FIG. 4, all 15/15 mice whichreceived a single AAV-GFP injection without NCs had exhibited antibodyreactivity against AAV, resulting in top ODs higher than normal serumcontrol (OD=0.227), while no mouse which received AAV co-administeredwith NCs exhibited a detectable level of antibodies to AAV. If only asingle AAV immunization was employed, levels of anti-AAV antibodiesstayed below the baseline at d21 in mice to which NCs wasco-administered with AAV, while being elevated in mice that received AAVwithout NCs (FIG. 5). At 33 days after a single injection, levels ofthese in untreated mice were still moderately growing, while inNCs-treated group 4 out of 5 mice had no detectable antibodies to AAV(FIG. 5).

If mice were boosted with AAV-GFP at day 21, then antibody levels inuntreated mice continued to grow significantly while being blunted inthose mice that received NCs only at boost (FIGS. 6 and 7).Interestingly, two mice in this latter group while being positive at d14(no treatment at prime) had their levels of antibodies to AAV fall belowbackground by d33 (FIG. 7). At the same time, 8/10 mice treated with NCsat prime had no detectable antibodies at d33 even after d21 boost (FIGS.6 and 7). Application of NCs at AAV boost may have had a minor effect inblocking generation of antibodies to AAV although at this point it wasnot statistically significant from no NCs treatment at boost (FIGS. 6and 7). Thus, NCs treatment at prime appears to be important forblocking the development of antibodies to AAV with its administration atlater time-points also being beneficial.

The results demonstrate the benefit of administering syntheticnanocarriers coupled to an immunosuppressant in conjunction with a viraltransfer vector for reducing antibody responses against the viraltransfer vector. Such benefits were seen with concomitant administrationof synthetic nanocarriers coupled to an immunosuppressant in conjunctionwith a viral transfer vector encoding a protein for expression.Accordingly, protocols for reducing anti-viral transfer vector antibodyresponses are hereinabove exemplified.

Example 12 Synthetic Nanocarriers Comprising Rapamycin Materials

PLA with an inherent viscosity of 0.41 dL/g was purchased from LakeshoreBiomaterials (756 Tom Martin Drive, Birmingham, Ala. 35211), productcode 100 DL 4A.

PLA-PEG-OMe block co-polymer with a methyl ether terminated PEG block ofapproximately 5,000 Da and an overall inherent viscosity of 0.50 DL/gwas purchased from Lakeshore Biomaterials (756 Tom Martin Drive,Birmingham, Ala. 35211), product code 100 DL mPEG 5000 5CE.

Rapamycin was purchased from Concord Biotech Limited (1482-1486 TrasadRoad, Dholka 382225, Ahmedabad India), product code SIROLIMUS.

Sorbitan monopalmitate was purchased from Sigma-Aldrich (3050 SpruceSt., St. Louis, Mo. 63103), product code 388920.

EMPROVE® Polyvinyl Alcohol (PVA) 4-88, USP (85-89% hydrolyzed, viscosityof 3.4-4.6 mPa·S) was purchased from EMD Chemicals Inc. (480 SouthDemocrat Road Gibbstown, N.J. 08027), product code 1.41350.

Dulbecco's phosphate buffered saline 1× (DPBS) was purchased from Lonza(Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland), product code17-512Q.

Method

Solutions were prepared as follows:

Solution 1:

A polymer, rapamycin, and sorbitan monopalmitate mixture was prepared bydissolving PLA at 37.5 mg/mL, PLA-PEG-Ome at 12.5 mg/mL, rapamycin at 8mg/mL, and sorbitan monopalmitate at 2.5 in dichloromethane.

Solution 2:

Polyvinyl alcohol was prepared at 50 mg/mL in 100 mM pH 8 phosphatebuffer.

An O/W emulsion was prepared by combining Solution 1 (1.0 mL) andSolution 2 (3 mL) in a small glass pressure tube, vortex mixed for 10seconds. The formulation was then homogenized by sonication at 30%amplitude for 1 minute. The emulsion was then added to an open beakercontaining DPBS (30 mL). A second O/W emulsion was prepared using thesame materials and method as above and then added to the same beakercontaining the first emulsion and DPBS. The combined emulsion was thenstirred at room temperature for 2 hours to allow the dichloromethane toevaporate and for the nanocarriers to form. A portion of thenanocarriers was washed by transferring the nanocarrier suspension to acentrifuge tube and centrifuging at 75,600×g and 4° C. for 50 minutes,removing the supernatant, and re-suspending the pellet in DPBScontaining 0.25% w/v PVA. The wash procedure was repeated and then thepellet was re-suspended in DPBS containing 0.25% w/v PVA to achieve ananocarrier suspension having a nominal concentration of 10 mg/mL on apolymer basis. The nanocarrier suspension was then filtered using a 0.22μm PES membrane syringe filter (Millipore part number SLGP033RB). Thefiltered nanocarrier suspension was then stored at −20° C.

Nanocarrier size was determined by dynamic light scattering. The amountof rapamycin in the nanocarrier was determined by HPLC analysis. Thetotal dry-nanocarrier mass per mL of suspension was determined by agravimetric method.

Effective Diameter Rapamycin Content Nanocarrier Conc (nm) (% w/w)(mg/mL) 150 11.5 11.1

Example 13 Single Administration of a Viral Transfer Vector with a GeneTherapy Transgene Induces Anti-Vector Antibody Responses that can beInhibited by Concomitant Administration with Synthetic NanocarriersCoupled to Immunosuppressant

A single intravenous (i.v.) administration of adeno-associated virusencoding a recombinant green fluorescent protein (AAV-GFP) (Virovek,Hayward, Calif.) under a CMV promoter led to an anti-AAV antibodyresponse that was inhibited by concomitant treatment withnanocarrier-encapsulated immunosuppressant (produced according toExample 12).

Experimental Methods

Male C57BL/6 mice were used (5-15 mice/group). Animals were injectedi.v. with 200 μL of AAV8-GFP or an admixture of AAV8-GFP+NCs, a PLGAnanocarrier containing rapamycin (see Table 2 below). On day 14 aftertreatment, all animals were bled and their sera analyzed for antibodiesagainst AAV8 by ELISA. Briefly, 96-well plates were coated with 50 μL ofAAV8 at 2×10⁹ vector genomes (vg)/mL in carbonate buffer for 92 hours,and then blocked for 2 hours with 300 μL of casein. Samples were addedat a 1:40 dilution in 50 μL of casein, and incubated for 2 hours at roomtemperature (RT). Horse radish peroxidase-conjugated rabbit anti-mouseIgG (Jackson ImmunoResearch, West Grove, Pa., 315-035-008) was used as asecondary antibody (0.5 μg/mL, 1 hour) and then TMB substrate was added(10 min) followed by the stop solution. Plates were then read at awavelength of 450 nm with a subtraction of background at 570 nm. Mousemonoclonal anti-AAV8 antibody (Fitzgerald, Acton, Mass., 10R-2136)served as a positive control.

Amounts of AAV-GFP:

1×10¹⁰ viral genomes (vg) at day 0 (prime) and 5×10¹⁰ vg at day 21(boost).

Amounts of Nanocarrier-Encapsulated Rapamycin (Rapa) Used:

50 μg of nanocarrier-entrapped rapamycin.

TABLE 2 Experimental Groups Gr. # Immunization, i.v. day 0 NCs (i.v. day0) 1 AAV-GFP (1 × 10¹⁰ VG) None 2 AAV-GFP (1 × 10¹⁰ VG) 50 μg of Rapa

Mice were bled at d14 and their sera tested for the presence ofantibodies to AAV8. At this point, all mice had been injected withAAV8-GFP once with or without co-administration of the nanocarriers (15mice each). As seen in FIG. 8, all mice which received a single AAV-GFPinjection without nanocarriers had exhibited antibody reactivity againstAAV8, resulting in antibody levels higher than the normal serum control(OD=0.227), while mice which received AAV8 co-administered with NCsexhibited little or no detectable levels of antibodies to AAV8. Levelsof anti-AAV8 antibodies stayed at or below the baseline at d21 in theNCs treated group (n=5), while being elevated in mice that receivedAAV8-GFP without NCs (FIG. 9). At 33 days after a single injection ofAAV8-GFP, anti-AAV8 antibody levels in untreated mice continued toincrease moderately, while in the NCs-treated group 4 out of 5 mice hadno detectable antibodies to AAV (FIG. 9).

The results demonstrate the benefit of administering syntheticnanocarriers coupled to an immunosuppressant in conjunction with a viraltransfer vector for reducing antibody responses against the viraltransfer vector. Such benefits were seen with concomitant administrationof synthetic nanocarriers coupled to an immunosuppressant in conjunctionwith an viral transfer vector comprising a transgene encoding a proteinfor expression. Accordingly, protocols for reducing anti-viral transfervector antibody responses are herein exemplified.

Example 14 Concomitant Administration of a Viral Transfer Vector with aGene Therapy Transgene with Synthetic Nanocarriers Coupled toImmunosuppressant Inhibits the Anti-AAV Antibody Response ExperimentalMethods

Male C57BL/6 mice were used (5 mice/group). Animals were injected with200 μL of AAV8-GFP (Virovek, Hayward, Calif.) or an admixture ofAAV8-GFP+NCs (as produced in Example 12) on day 0 and/or day 21 asindicated in Table 3. Sera were collected on day 0 and 33 and analyzedfor anti-AAV8 antibody levels by ELISA as described above.

Amounts of AAV-GFP:

1×10¹⁰ viral genomes (vg) at d0 prime, 5×10¹⁰ vg at d21 boost.

Amounts of Nanocarrier-Encapsulated Immunosuppressant (Rapamycin orRapa) Used:

50 μg of nanocarrier-entrapped Rapa at either prime (gr. 2 and 4) orboost (gr. 3 and 4).

TABLE 3 Experimental Groups Day 0 Day 21 Gr. Viral transfer Viraltransfer # vector (i.v.) NCs (i.v.) vector (i.v.) NCs (i.v.) 1 AAV-GFPNone AAV-GFP None (1 × 10¹⁰ vg) (5 × 10¹⁰ vg) 2 AAV-GFP None AAV-GFP 50μg of Rapa (1 × 10¹⁰ vg) (5 × 10¹⁰ vg) 3 AAV-GFP 50 μg of Rapa AAV-GFPNone (1 × 10¹⁰ vg) (5 × 10¹⁰ vg) 4 AAV-GFP 50 μg of Rapa AAV-GFP 50 μgof Rapa (1 × 10¹⁰ vg) (5 × 10¹⁰ vg)

Results

Mice injected with AAV8-GFP on day 0 in the absence of NCs showed arobust anti-AAV8 antibody response that increased significantly afterthe second injection of AAV8-GFP on day 21 (FIG. 10). However if NCs wasconcomitantly administered with the AAV8-GFP on day 21, the antibodyresponse on average was significantly blunted. Interestingly, two micein this latter group which were antibody positive on d14 had nodetectable levels of antibodies to AAV8 on d33 (FIG. 10). Howeveranti-AAV8 antibody titers increased in 2 other mice. In contrast, NCsconcomitantly administered at the time of the first AAV8-GFP injection(day 0) completely inhibited the anti-AAV8 antibody response at day 14.The anti-AAV8 antibody was also inhibited in 4 of 5 mice at day 33 aftera second administration of AAV8-GFP alone on day 21. Concomitantadministration of NCs at both day 0 and 21 showed a similar trend. Thus,NCs treatment at the time of the first administration of AAV isimportant for blocking the development of antibodies to AAV. Additionaladministration of NCs upon repeat dosing of AAV may be potentiallybeneficial.

The results demonstrate the benefit of administering syntheticnanocarriers coupled to an immunosuppressant in conjunction with a viraltransfer vector for reducing antibody responses against the viraltransfer vector. Such benefits were seen with concomitant administrationof synthetic nanocarriers coupled to an immunosuppressant in conjunctionwith a viral transfer vector encoding a protein for expression.Accordingly, protocols for reducing anti-viral transfer vector antibodyresponses are herein exemplified.

Example 15 Therapeutic Administration of Synthetic Nanocarriers Coupledto Immunosuppressant Enhances the Maintenance of Transgene ExpressionUpon Repeat Dosing of a Viral Transfer Vector

Two successive intravenous (i.v.) inoculations of adeno-associated virusencoding recombinant green fluorescent protein (AAV8-GFP) (Virovek,Hayward, Calif.) led to higher GFP expression in liver cells in vivo ifnanocarrier-encapsulated immunosuppressant (NCs) (as produced in Example12) was co-injected at the time of a repeat administration of a viraltransfer vector encoding a protein for expression.

Experimental Methods

Male C57BL/6 mice were used (5 mice/group). Animals were injected with200 μL of AAV8-GFP in the absence of NCs on day 0. One group of animalsreceived no further treatment, while other groups received a second doseof AAV8-GFP on day 21 with or without concomitant administration of NCscarrying 50 μg rapamycin (see Table 4 below). At d33 after the firstinjection (12 days after the second injection for those groups that wereinjected twice) animals were sacrificed, their livers treated withcollagenase 4 (Worthington, Lakewood, N.J.), meshed and total cellsuspensions were analyzed by flow cytometry for GFP expression. Briefly,tissue was initially perfused with collagenase (100 U) and incubated at37° C. for 30 min. The collagenase supernatant was removed, and quenchedwith 2% FBS. Tissue samples were then cut into ˜2 mm squares, digested(collagenase, 400 U) with repeated agitation, filtered (nylon mesh),spun down (1,500 rpm), and pellets re-suspended in ice-cold 2% FBS.

Amounts of AAV-GFP:

1×10¹⁰ viral genomes (vg) at d0 and 5×10¹⁰ vg at d21 (groups 2 and 3only).

Amounts of Nanocarrier-Encapsulated Immunosuppressant (Rapamycin orRapa) Used:

50 μg of nanocarrier-entrapped Rapa at day 21 (gr. 3).

TABLE 4 Experimental Groups Day 0 Day 21 Gr. Viral transfer Viraltransfer # vector (i.v.) vector (i.v.) NCs (i.v.) 1 AAV-GFP None None (1× 10¹⁰ vg) 2 AAV-GFP AAV-GFP None (1 × 10¹⁰ vg) (5 × 10¹⁰ vg) 3 AAV-GFPAAV-GFP 50 μg of Rapa (1 × 10¹⁰ vg) (5 × 10¹⁰ vg)

Results

Statistically higher levels of GFP expression in the liver of AAV8-GFPtreated mice were seen if NCs was concomitantly administered with thesecond injection of AAV8-GFP on d21 compared to animals that received asecond injection of AAV8-GFP in the absence of NCs (FIG. 11). The levelof GFP expression observed in mice that received a second injection ofAAV8-GFP plus NCs was similar to that observed in mice that receivedonly a single dose of AAV8-GFP on day 0. These results indicate that theco-administration of NCs at the time of the second dose of AAV8-GFP wasimportant to maintain expression of GFP, perhaps by inhibiting cytolyticT cells which could eliminate transduced liver cells expressing GFP.

The results demonstrate the benefit of administering syntheticnanocarriers coupled to an immunosuppressant in conjunction with a viraltransfer vector for maintaining expression of the vector transgene. Suchbenefits were seen with concomitant administration of syntheticnanocarriers coupled to an immunosuppressant in conjunction with a viraltransfer vector comprising a transgene encoding a protein forexpression.

Example 16 Concomitant Administration of a Synthetic NanocarriersCoupled to Immunosuppressant Enhances Transgene Expression ExperimentalMethods

Male C57BL/6 mice were used (5 mice/group). Animals were injected with200 μL of AAV-red fluorescence protein (RFP) (Virovek, Hayward, Calif.)on day 0 (groups 1-5) and/or day 21 (groups 1-4, 6) (see Table 5 below).NCs carrying 50 μg rapamycin was concomitantly administered on day 0(groups 2, 4) and/or day 21 (groups 3, 4). At d33 after the firstinjection (12 days after the second injection for those groups that wereinjected twice) animals were sacrificed, their livers treated withcollagenase 4 (Worthington, Lakewood, N.J.), meshed and total cellsuspensions were analyzed by flow cytometry for RFP expression. Briefly,tissue was initially perfused with collagenase (100 U) and incubated at37° C. for 30 min. The collagenase supernatant was removed and quenchedwith 2% FBS. Tissue samples were then cut into ˜2 mm squares, digested(collagenase, 400 U) with repeated agitation, filtered (nylon mesh),spun down (1,500 rpm), and pellets re-suspended in ice-cold 2% FBS.

TABLE 5 Experimental Groups Day 0 Day 21 Viral transfer Viral transferGr. # vector (i.v.) NCs (i.v.) vector (i.v.) NCs (i.v.) 1 AAV-RFP NoneAAV-RFP None 2 AAV-RFP 50 μg of Rapa AAV-RFP None 3 AAV-RFP None AAV-RFP50 μg of Rapa 4 AAV-RFP 50 μg of Rapa AAV-RFP 50 μg of Rapa 5 AAV-RFPNone None None 6 None None AAV-RFP None

Results

Animals administered one or two injections of AAV8-RFP in the absence ofNCs showed similar low levels of RFP expression at day 33 (FIG. 12).Mice treated with NCs concomitantly at the time of the first injectionof AAV8-RFP showed a trend towards increased expression of RFP that wasnot statistically significant. In contrast, mice that were treatedconcomitantly with NCs at the time of the second injection of AAV8-RFP(day 21) showed a statistically significant increase in RFP expression.Mice that were treated with NCs at both day 0 and day 21 also showed asignificant increase in RFP expression compared to control animals thatreceived AAV8-RFP on days 0 and 21 in the absence of NCs.

The results demonstrate the benefit of administering syntheticnanocarriers coupled to an immunosuppressant in conjunction with a viraltransfer vector for enhancing expression of the vector transgene. Suchbenefits were seen with concomitant administration of syntheticnanocarriers coupled to an immunosuppressant in conjunction with a viraltransfer vector comprising a transgene encoding a protein forexpression.

Example 17 Administration of a Viral Transfer Vector with a Gene TherapyTransgene with Synthetic Nanocarriers Coupled to ImmunosuppressantInhibits CD8+ T Cell Activation

Two successive intravenous (i.v.) inoculations of adeno-associated virusexpressing recombinant green fluorescent protein (AAV-GFP) (Virovek,Hayward, Calif.) led to lower cytolytic T cell (CTL) activity againstAAV capsid protein and GFP in vivo if nanocarrier-encapsulatedimmunosuppressant (NCs) was co-injected with both AAV8-GFP injections.

Experimental Methods

Male C57BL/6 mice were used (3 or 6 mice/group). Animals were injectedwith 200 μL of AAV-GFP or an admixture of AAV-GFP+NCs on day 0 and day17 or 21 (see Table 6 below). Groups injected at days 0 and 21 (n=3 miceper group) were assayed for antigen-specific CTL activity at 28 daysafter the first injection (7 days after the second injection). Briefly,splenocytes from syngeneic naïve mice were labeled with either 0.5 μM,or 5 μM CFSE, resulting in CFSE^(low) and CFSE^(high) cell populations,respectively. CFSE^(high) cells were incubated with 1 μg/mL dominant MHCclass I-binding peptide from AAV capsid (sequence NSLANPGIA, amino acids517-525) and dominant MHC class I peptide from GFP (HYLSTQSAL, aa200-208) at 37° C. for 1 h, while CFSE^(low) cells were incubated inmedium alone. The control CFSE^(low) cells and peptide-pulsedCFSE^(high) target cells were mixed in a 1:1 ratio (2.0×10⁷ cells total)and injected i.v. Eighteen hours after the injection of labeled cells,spleens were harvested, processed and analyzed by flow cytometry.Specific cytotoxicity was calculated based on a control ratio ofrecovery (RR) in naïve mice:(percentage of CFSE^(low) cells)/(percentageof CFSE^(high) cells). Percent specific lysis (%)=100×[1−(RR of cellsfrom naive mice/RR of cells from immunized mice) or100×[1−(RR_(naive)/RR_(imm))].

Groups injected at days 0 and 17 (n=6 mice per group) were assayed forantigen-specific IFN-γ production on d25 after the first injection (7days after the second injection). Briefly, splenocytes were isolated,plated in wells with pre-absorbed anti-IFN-γ antibody and re-stimulatedwith AAV capsid or GFP peptides (1 μg/mL) for 7 days in vitro. ELISpotswere developed by biotinylated anti-IFN-γ antibody and streptavidin-HRP,and spots were counted. Nonspecific background was subtracted.

Amounts of AAV-GFP:

1×10¹⁰ viral genomes (vg) at d0 prime, 5×10¹⁰ vg at d21 boost.

Amounts of Nanocarrier-Encapsulated Immunosuppressant (Rapamycin orRapa) Used:

50 μg of nanocarrier-entrapped Rapa at both prime and boost (gr. 2).

TABLE 6 Experimental Groups First Injection i.v. (Day 0) SecondInjection i.v. (day 17 or 21) Gr. # AAV8-GFP NCs Challenge NCs 1 1 ×10¹⁰ vg None 5 × 10¹⁰ vg None 2 1 × 10¹⁰ vg 50 μg rapamycin 5 × 10¹⁰ vg50 μg rapamycinResults Animals concomitantly treated with NCs showed lower levels of invivo CTL activity against targets cells pulsed with a combination of AAVcapsid and GFP dominant MHC class I peptides (FIG. 13). Similarly, miceconcomitantly treated with NCs showed a significant reduction inantigen-specific IFN-γ-producing cells compared to the non-NCs-treatedgroup (FIGS. 14 and 15). In particular, 4/6 mice demonstrated a recallresponse to the AAV capsid protein at 250,000 cells per well density,while no (0/6) mice responded to this peptide in the NCs-treated group(FIG. 14, p<0.05). Moreover, 3/6 mice in the AAV8-GFP-immunized groupshowed a response to an immunodominant GFP peptide, while no mice (0/6)responded to this peptide in the NCs-treated group (FIG. 15, p=0.01).

Collectively, it appeared that co-administration of AAV-GFP and NCs atprime and boost results in suppression of cytotoxic T cell responsesagainst viral capsid and transgenic proteins.

Example 18 Administration of Viral Transfer Vector with a Gene TherapyTransgene with Synthetic Nanocarriers Comprising ImmunosuppressantExperimental Methods

Male C57BL/6 mice were used (5 mice/group). Animals were injected i.v.with 10¹⁰ vg of rAAV2/8-luciferase (rAAV2/8-Luc)) (produced in a mannersimilar to the methods provided herein such as in Example 21 or 22) orrAAV2/8-Luc+synthetic nanocarriers containing 100 μg rapamycin (NCs) onDay 0 (see Table 7 below). On day 14, all animals received an i.v.injection of 10¹⁰ vg of rAAV2/8 encoding human factor IX (hFIX)(rAAV2/8-hFIX)) (produced in a manner similar to the methods providedherein such as in Example 21 or 22). Sera were collected at various timepoints and assayed for anti-AAV antibody levels and hFIX protein levelsby ELISA.

FIG. 16 illustrates the protocol and timing of administration ofsynthetic nanocarriers comprising an immunosuppressant. Synthetic PLGAnanocarriers containing 100 μg rapamycin (NCs) or control emptynanoparticles (Empty NP) were administered i.v. concomitantly withrAAV2/8-Luc vector (10¹⁰ vg) on Day 0 (N=5/group). All groups receivedan injection (i.v.) of rAAV2/8 vector encoding human coagulation factorIX (hFIX) on Day 14. The data show that a single administration ofsynthetic nanocarriers comprising immunosuppressant concomitantlyadministered with AAV8-Luc can prevent or delay the onset of anti-AAV8antibodies (FIG. 17). Importantly, the concomitant administration of NCswith rAAV2/8-luciferase inhibited anti-AAV8 antibody formationsufficiently to enable efficient expression of hFIX from therAAV2/8-hFIX administered on day 14. In contrast, animals treated withempty NP developed anti-AAV8 antibodies which prevented efficientexpression of hFIX from the rAAV2/8-hFIX vector administered on day 14.These data indicate that concomitant administration of syntheticnanocarriers comprising immunosuppressant at the time of the firstapplication of AAV enables efficacious repeat dosing of the sameserotype of AAV.

TABLE 7 Treatment Groups Groups Nanocarriers rAAV2/8-Luc rAAV2/8-FIX N 1NCs, D 0 D 0 D 14 5 2 Empty NP, D 0 D 0 D 14 5

Example 19 Multiple Administrations of Viral Transfer Vector with a GeneTherapy Transgene with Synthetic Nanocarriers ComprisingImmunosuppressant

The experimental design is shown in FIG. 18. Male C57BL/6 mice were used(5 mice/group). Synthetic nanocarriers containing rapamycin (NCs) (100μg rapamycin) were administered i.v. concomitantly with rAAV2/8-Lucvector) (produced in a manner similar to the methods provided hereinsuch as in Example 21 or 22) (1×10¹¹ vg) on day 0 (N=5/group) (Table 8).Mice were then challenged with rAAV2/8-hFIX) (produced in a mannersimilar to the methods provided herein such as in Example 21 or 22)concomitantly administered with synthetic nanocarriers containingrapamycin (100 μg rapamycin) on day 21. The control group received emptyNP instead of NCs on days 0 and 21.

TABLE 8 Treatment Groups rAAV2/8-Luc rAAV2/8-FIX Groups Nanoparticles(10¹¹ vg) (10¹¹ vg) N 1 NCs, d 0 21 d 0 d 21 5 2 Empty NP, d 0 21 d 0 d21 5

The results showed that concomitant administration of syntheticnanocarrier injections with both the first (rAAV2/8-Luc) and second(rAAV2/8-hFIX) injections of viral transfer vector inhibited theanti-AAV8 antibody response (FIG. 19, left panel) and reduced the titerof neutralizing antibodies to AAV8 (Table 9). The inhibition of theanti-AAV8 antibodies enabled higher levels of AAV2/8-hFIX vector copynumbers (FIG. 19, middle panel), which in turn provided for robustexpression of the hFIX transgene (FIG. 19, right panel). Note that inthe control group treated with empty nanoparticles, several animals hadlow levels of antibodies at day 20. Two of these animals had anintermediate level of vector copy numbers and some expression of hFIX inresponse to administration of rAAV2/8-hFIX on day 21. However three ofthe control animals showed very low vector copy numbers and nodetectable levels of FIX expression.

Accordingly, it was demonstrated that multiple administrations ofsynthetic nanocarriers comprising immunosuppressant can completelyprevent the induction of antigen-specific anti-AAV8 antibodies, allowingfor high levels of transgene expression upon a second injection of AAV.

TABLE 9 Neutralizing anti-AAV antibody titers AAV8 Neutralizing AntibodyTiter NCs Empty NP Animal # Day 20 Day 41 Day 20 Day 41 1     1:3.161:31.6 1:31.6 1:1000 2 <1:1 1:31.6 1:31.6 1:316  3 <1:1 1:31.6 1:31.61:316  4 <1:1 1:31.6 1:31.6 1:1000 5 <1:1 1:31.6 1:31.6 1:1000

Example 20 Antigen Specificity

The experimental design is shown in FIG. 20. Synthetic nanocarrierscomprising immunosuppressant (100 μg rapamycin) or control emptynanoparticles were administered i.v. concurrently with rAAV2/8-Lucvector) (produced in a manner similar to the methods provided hereinsuch as in Example 21 or 22) (1×10¹¹ vg/mouse) on Day 0. On day 21 micereceived either an i.v. injection of rAAV5-hFIX) (produced in a mannersimilar to the methods provided herein such as in Example 21 or 22)(1×10¹¹ vg/mouse) or an i.m. injection of human Factor IX (hFIX) proteinemulsified in complete Freund's adjuvant (CFA) (Table 10).

TABLE 10 Treatment Groups FIX rAAV2/8-Luc rAAV5-hFIX protein GroupsNanoparticles (10¹¹ vg) (10¹¹ vg) CFA N 1 NCs, d 0 d 0 d 21 — 5 2 EmptyNP, d 0 d 0 d 21 — 5 3 NCs, d 0 d 0 — d 21 5 4 Empty NP, d 0 d 0 — d 215

The results showed that concomitant i.v. administration of syntheticnanocarrier carrying rapamycin with an rAAV2/8 vector (AAV2/8-Luc) onday 0 did not have a profound impact on the antibody response to an AAV5vector (AAV5-hFIX) administered on day 21 (FIG. 21, left panel). Theanimals treated with the NCs containing rapamycin had a short delay inthe anti-AAV5 antibody response compared to mice treated with empty NP,perhaps because of the presence of B cells in the the empty NP-treatedmice that were primed against AAV8 and crossreactive to AAV5. Howeverthe anti-AAV5 antibody response of the NCs-treated mice rapidlyparallels the anti-AAV5 antibody response of the empty NP-treated group.

In contrast, animals that received AAV2/8-FIX on day 21 showed little orno anti-AAV8 antibodies. These data indicate that the effect of the NCstreatment on inhibiting anti-AAV antibody responses were specific to AAVserotype with which it was co-administered (i.e., AAV8) and does notrender the mice chronically immunosuppressed. Similarly, miceconcomitantly treated with NCs and rAAV2/8-Luc on day 0 showed a robustresponse to immunization with recombinant hFIX protein in completeFreund's adjuvant (CFA) on day 21 (FIG. 21, right panel). The anti-hFIXantibody response was indistinguishable from that of mice that weretreated with empty NP instead of NCs on day 0. Accordingly, it wasdemonstrated that concomitant administration of synthetic nanocarrierscomprising immunosuppressant with AAV does not result in chronicimmunosuppression.

Example 21 AAV5 Transfer Vector with a Gene Therapy Transgene(Prophetic)

ART-I02 is produced as described previously (Matsushita T, et al. GeneTher. 1998; 5: 938-945). The plasmid encodes any one of the transgenesprovided herein under the control of the NF-κB promoter and a humangrowth hormone polyadenylation signal. The gene of interest may also beunder the control of the cytomegalovirus (CMV) promoter. The transgenecassette is flanked by AAV-2 inverted terminal repeats and is packagedin capsid from AAV5 as described in Gao G P, et al. Proc Natl Acad SciUSA. 2002; 99: 11854-11859. The vector is purified by combinedchromatography and cesium chloride density gradient centrifugation,resulting in empty capsid-free fractions. Vector titers can bedetermined by qPCR using specific primers and probe. Similarly, as anexample, a rAAV5 vector can be produced coding for Firefly Luciferase.

Example 22 AAV2/8 and AAV2/5 Transfer Vector with a Gene TherapyTransgene (Prophetic)

An scAAV backbone plasmid is constructed by ligating MscIBsaI andBsaITsp45I fragments from AAV2-HCR-hAAT-FIX to the simian virus 40 latepolyA (SV40 LpA). The resulting plasmid contains the modified AAV2backbone with an intact 5′ terminal resolution site (trs) and a deleted3′ trs. The LP1 enhancer/promoter can be constructed using standardpolymerase chain reaction (PCR) methods with amplification ofconsecutive segments of the human apolipoprotein hepatic control region(HCR), the human alpha1antitrypsin (hAAT) gene promoter including the 5′untranslated region and cloned upstream of a modified SV40 small tantigen intron (SV40 intron) modified at positions 4582 (g to c), 4580(g to c), 4578 (a to c), and 4561 (a to t) into the modified AAV2backbone. The wild-type hFIX cDNA, or other cDNA of interest, withoutthe 3′ untranslated region (UTR) regions is PCR amplified fromAAV-HCR-hAAT-hFIX and inserted downstream of the modified SV40 intron tomake scAAV-LP1-hFIX. A codon-optimized hFIX is generated using codonsmost frequently found in highly expressed eukaryotic genes, synthesizedas oligonucleotides, and subsequently assembled by ligation, PCRamplified, and sequenced prior to cloning into the AAVLP1 backbone tocreate sc-AAV-LP1-hFIXco. ss and scAAV vectors are made by theadenovirus-free transient transfection method.

AAV5-pseudotyped vector particles are generated using a chimeric AAV2Rep-5Cap packaging plasmid called pLT-RCO3, which is based on XX2 andpAAV5-2. Additionally, AAV8-pseudotyped vectors are made using thepackaging plasmid pAAV8-2. AAV2/5 and 2/8 vectors are purified by theion exchange chromatography method. Vector genome (vg) titers can bedetermined by quantitative slotblot using supercoiled plasmid DNA asstandards. Such a viral vector can comprise any one of the transgenes asprovided herein.

Example 23 AAV8 Transfer Vector with a Gene Therapy Transgene(Prophetic)

A mouse genomic Alb segment (90474003-90476720 in NCBI referencesequence: NC_(—)000071.6) can be PCR-amplified and inserted between AAV2inverted terminal repeats into BsrGI and SpeI restriction sites in amodified pTRUF backbone. An optimized P2A coding sequence preceded by alinker coding sequence (glycine-serine-glycine) and followed by anNheIrestriction site can be into the Bpu10I restriction site. Acodon-optimized F9 coding sequence can be inserted into the NheI site toget pAB269 that can serve in the construction of the rAAV8 vector. Toconstruct the inverse control, an internal segment from the BsiWIrestriction site to the NheI restriction site can be amplified usingappropriate PCR primers. Final rAAV production plasmids can be generatedusing an EndoFree Plasmid Megaprep Kit (Qiagen).

rAAV8 vectors can be produced as described in Grimm, et al., J. Virol.80, 426-439 (2006) using a Ca₃(PO₄)₂ transfection protocol followed byCsCl gradient purification. Vectors can be titred by quantitative dotblot.

As described in Barzel, et al., 364, Nature, Vol. 517, 2015,amelioration of the bleeding diathesis in haemophilia B mice wasdemonstrated using such vectors as described above. In particular, thevectors achieved integration into the albumin alleles in hepatocytes. F9was produced from on-target integration, and ribosomal skipping washighly efficient. Stable F9 plasma levels were obtained, and treatedF9-deficient mice had normal coagulation times.

Example 24 AAV9 Transfer Vector with a Gene Therapy Transgene(Prophetic)

Adenoviral constructs using a “first-generation” E1/E3-deletedreplication-deficient adenovirus can be produced as described in Kypson,et al. J Thorac Cardiovasc Surg. 1998 and Akhter, et al. Proc Natl AcadSci USA. 1997; 94:12100-12105. The b₂AR construct (Adeno-b₂AR) and atransgene can be driven by an appropriate promoter. Large-scalepreparations of adenoviruses can be purified from infected Epstein-Barrnuclear antigen-transfected 293 cells.

As described in Shah et al., Circulation. 2000; 101:408-414, rabbitsthat underwent percutaneous subselective catheterization of either theleft or right coronary artery and infusion of adenoviral vectors such asthose produced as above containing a marker transgene expressed thetransgene in a chamber-specific manner. In addition, it was concludedthat percutaneous adenovirus-mediated intracoronary delivery of atherapeutic transgene is feasible, and that acute global leftventricular function can be enhanced.

Example 25 Lentiviral Transfer Vector with a Gene Therapy Transgene(Prophetic)

The following can be prepared: a lentiviral expression plasmidcontaining a packaging sequence and a transgene inserted between thelentiviral LTRs to allow target cell integration; a packaging plasmid,encoding the pol, gag, rev and tat viral genes and containing therev-response element; and a pseudotyping plasmid, encoding a protein, ofa virus envelope gene. HEK 293T cells can be transfected by theforegoing. After transfection of HEK 293T cells, the lentiviral vectorscan be obtained from the cell supernatant which contains recombinantlentiviral vectors.

Example 26 HIV Lentiviral Transfer Vector with a Gene Therapy Transgene(Prophetic)

An HIV lentiviral transfer vector is prepared according the methods ofU.S. Publication No. 20150056696. An hPEDF CDS fragment is PCR amplifiedfrom cDNA of the human Retinal pigment epithelium cell strain ARPE-19(American Type Culture Collection, ATCC) as a template and usingappropriate primers. An alternative fragment can be similarly obtainedfor any one of the proteins described herein. The hPEDF fragment isobtained by gel recovery and ligated into the pLenti6.3/V5-TOPO® vector(Invitrogen) by TA cloning procedure following the manufacturer'sinstruction. The sequence of the ligated hPEDF fragment can be verifiedby sequencing.

Example 27 SIV Lentiviral Transfer Vector with a Gene Therapy Transgene(Prophetic)

An SIV lentiviral transfer vector is prepared according the methods ofU.S. Publication No. 20150056696. A SIV gene transfer vector, apackaging vector, a rev expression vector, and a VSV-G expression vectorare obtained, and an hPEDF fragment is introduced into the gene transfervector. An alternative fragment can be similarly obtained for any one ofthe proteins described herein for introduction into the gene transfervector.

The cell line 293T cells derived from human fetal kidney cells areseeded at a cell density of approximate 1×10⁷ cells per plastic Petridish having the diameter of 15 cm (cell density of 70-80% next day) andcultured in 20 ml of D-MEM culture medium (Gibco BRL) supplemented with10% fetal bovine serum for 24 hrs. After 24 h of cultivation, theculture medium is replaced with 10 ml of OPTI-MEM culture medium (GibcoBRL).

For one petri dish, 10 μg of the gene transfer vector, 5 μg of thepackaging vector, 2 μg of the rev expression vector and 2 μg of VSV-Gexpression vector are dissolved in 1.5 ml of OPTI-MEM medium, and 40 μlof PLUS Reagent reagent (Invitro Co.) is added. The resulting mixture isstirred and left at room temperature for 15 min. A dilute solution isobtained by diluting 60 μl of LIPOFECT AMINE Reagent with 1.5 ml ofOPTI-MEM medium; the resulting mixture is stirred and left at roomtemperature for 15 min. The resulting DNA-complex is dropped onto thecells in the above-described Petri dish. The Petri dish is shakencarefully to achieve uniform mixing, and then incubated. 13 ml of D-MEMmedium comprising 20% of fetal bovine serum is added. Supernatant isrecovered.

Example 28 HSV Transfer Vector with a Gene Therapy Transgene (Prophetic)

An HSV transfer vector is prepared according the methods of U.S.Publication No. 20090186003. HSV-1 (F) strain is a low passage clinicalisolate used as the prototype HSV-1 strain. M002, which expresses murineinterleukin 12 (mIL-12) under the transcriptional control of the murineearly-growth response-1 promoter (Egr-1), is constructed. Alternatively,similar constructs may be prepared encoding any one of the proteinsdescribed herein under the control of an appropriate promoter. Theplasmids containing the p40 and p35 subunits of mIL-12 inpBluescript-SK+ (Stratagene) are obtained. The p40 subunit is removed bydigestion with HindIII (5′ end) and BamHI (3′ end) and the p35 subunitis removed by digestion with NcoI (5′ end) and EcoRI (3′ end). Theinternal ribosome entry site, or IRES, sequence is amplified from vectorpCITE-4a+ (Novagen, Madison, Wis.) using polymerase chain reaction (PCR)and appropriate primers. Plasmid pBS-IL12 is constructed by three-wayligation of the murine p40, murine p35 and IRES sequences into HindIIIand EcoRI sites of pBS-SK+ such that the IRES sequence separates the p40and p35 coding sequences.

A HSV shuttle plasmid pRB4878 can be prepared as previously described(Andreansky et al. (1998) Gene Ther. 5, 121-130). Plasmid 4878-IL12 isconstructed as follows: pBS-mIL-12 is digested with XhoI and SpeI toremove a 2.2 kb fragment containing the entire IL-12 subunit codingregions, including the IRES, ends filled in using the Klenow fragment,and ligated into a blunted KpnI site located between the Egr-1 promoterand hepatitis B virus polyA sequences within pRB4878. M001 (tk−) andM002 (tk repaired at native locus) are constructed via homologousrecombination as described previously (Andreansky et al. (1998) GeneTher. 5, 121-130). Two tk-repaired viruses M002.29 and M002.211, areconfirmed by Southern blot hybridization of restriction enzyme-digestedviral DNAs which are electrophoretically separated on a 1% agarose,1×TPE gel and transferred to a Zeta-Probe membrane (Bio-Rad). The blotis hybridized with the appropriate DNA probe labeled with alkalinephosphatase using the Gene Images AlkPhos Direct DNA labeling system(Amersham-Pharmacia Biotech, Piscataway, N.J.). IL-12 production isdemonstrated by enzyme-linked immunosorbent assay (ELISA).

Example 29 Viral Transfer Vector with a CRISPR/Cas-9 Transgene(Prophetic)

Any one of the viral vectors described herein, such as in the aboveExamples, may be used to produce a viral transfer vector with a geneediting transgene. Alternatively, and as an example, the followingprovides a method for producing a viral transfer vector with a geneediting transgene that encodes Cas9, such as Cas9 wild-type (Type II).

HEK293T cells can be cultured in DMEM medium (Life Technologies,Darmstadt, Germany) containing 10% fetal bovine serum (Sigma, Steinheim,Germany), 100 U/mL penicillin and 100 μg/mL streptomycin (LifeTechnologies). Huh7 and Hep56D cell media can additionally contain 1%non-essential amino acids (Life Technologies). Jurkat cells can be grownin RPMI 1640 medium (GE Healthcare, Pasching, Austria) containing 10%fetal bovine serum (Sigma), 100 U/mL penicillin, 100 μg/mL streptomycin,and 2 mM L-glutamine (all Life Technologies). All cell lines can becultured at 37° C. and 5% CO2. For large-scale AAV vector production,HEK293T cells can be seeded in ten 15 cm2 dishes (4×106 cells per dish).Two days later, they can be triple-transfected with (i) the AAV vectorplasmid (encoding gRNA and/or Cas9), (ii) an AAV helper plasmid carryingAAV rep and cap genes, and (iii) an adenoviral plasmid providing helperfunctions for AAV production.

The AAV cap gene can be either derived from the synthetic isolate AAV-DJ(Grimm, et al., J. Virol. 2008, 82, 5887-5911) or from a new variantAAVrh10A2. Briefly, AAVrh10A2 can be created through insertion of aseven amino acid long peptide into an exposed region of the capsid ofAAV serotype rh10. Further details on AAV production plasmids andprotocols can be performed as reported in Börner, et al., Nucleic AcidsRes. 2013, 41, e199 and Grimm, Methods 2002, 28, 146-157. To generatesmall-scale AAV stocks, 2×105 HEK293T cells per well can be seeded in6-well plates and the next day triple-transfected with theaforementioned plasmids. Three days later, the cells can be scraped offinto the media, collected via 10 min centrifugation at 1500 rpm,resuspended in 300 μL 1×PBS (Life Technologies) and subjected to threefreeze-thaw cycles in liquid nitrogen and at 37° C. A 10 mincentrifugation can be performed at 13,200 rpm to remove cell debris, andsupernatants containing viral transfer vector particles can be useddirectly in transduction experiments or frozen at −20° C.

For small-scale transfections and subsequent T7 assays, 2.8×104 HEK293Tor 1.2×104 Huh7 cells can be seeded per well in a 96-well plate and thenext day transfected using lipofectamine 2000 (Life Technologies)following the manufacturer's recommendations for this format (200 ng DNAand 0.5 μL lipofectamine 2000, each in 25 μL serum-free medium). The 200ng DNA can consist of an all-in-one Cas9/gRNA vector, or, in the case ofseparate Cas9 and gRNA constructs, of 100 ng of each. To obtain lysatesfor Western blotting, HEK293T cells can be transfected in 24-well plates(one well per lysate), using lipofectamine 2000 according to themanufacturer's recommendations for this format. In transductionexperiments, cells can be grown in 96-well plates and transduced witheither 10 μL non-purified AAV or with purified vector 1 day afterseeding. Following a three (transfections) to five (transductions) dayincubation, the cells can be lysed with DirectPCR Lysis Reagent Cell(PeqLab, Erlangen, Germany) supplemented with 0.2 μg/mL proteinase K(Roche, Mannheim, Germany) following the manufacturer's protocol.

As described in Senís, et al. Biotechnol. J. 2014, 9, 1402-1412,plasmids and vectors such as those above can achieve delivery of theCRISPR components—Cas9 and chimeric g(uide) RNA. In addition, it wasdemonstrated that Cas9 expression could be directed to or away fromhepatocytes, using a liver-specific promoter or a hepatic miRNA bindingsite, respectively. Further evidence was provided that such vectors canbe used for gene engineering in vivo. This was accomplished in theexemplified liver of adult mice.

Example 30 Viral Transfer Vector with a Cas9 Variant Transgene(Prophetic)

The methodology in Example 30 can also be used to produce a viraltransfer vector with a gene editing transgene, such as a transgeneencoding a Cas9 variant, such as any one of the Cas9 variants describedherein. Alternatively, any one of the other viral vectors describedherein, may be used instead to produce such a viral transfer vector. Anyone of the Cas9 variants provided can be encoded by any one of the geneediting transgenes provided herein.

To make Cas9 variants, the human codon-optimized streptococcus pyogenesCas9 nuclease with NLS and 3× FLAG tag (Addgene plasmid 43861) can beused as the wild-type Cas9 expression plasmid. PCR products of wild-typeCas9 expression plasmid as template with Cas9_Exp primers can beassembled with Gibson Assembly Cloning Kit (New England Biolabs) toconstruct Cas9 and FokI-dCas9 variants. Expression plasmids encoding asingle gRNA construct (gRNA G1 through G13) can also be cloned.

Example 31 Viral Transfer Vector with a Zinc Finger Nuclease Transgene(Prophetic)

Any one of the viral vectors described herein, such as in the aboveExamples, may be used to produce a viral transfer vector with a geneediting transgene that encodes a zinc finger nuclease. Alternatively,and as an example, the following provides a method for producing such aviral transfer vector with a gene editing transgene.

TRBC- and TRAC-ZFNs can be designed and assembled as described in Urnov,et al. Nature 435, 646-651 (2005). The recognition helices used can beas provided Provasi, et al., Nature Medicine, Vol. 18, No. 5, May 2012.Lentiviral vectors encoding TRBC- and TRAC-ZFNs can be generated fromthe HIV-derived self-inactivating transfer constructpCCLsin.cPPT.SFFV.eGFP.Wpre, which can be packaged by anintegrase-defective third generation packaging construct carrying theD64V mutation in the HIV integrase and pseudotyped by the VSV envelope.The Ad5/F35 adenoviral vectors can be generated on an E1-E3-deletedbackbone. The ZFNs targeting either the TRBC or TRAC gene can be linkedusing a 2A peptide sequence and cloned into the pAdEasy-1/F35 vectorunder the control of an appropriate promoter, and the Ad5/F35 virus foreach construct can be generated using TREx 293T cells. Lentiviralvectors encoding both WT1-specific TCR chains and single α21 or β21WT1-specific TCR chains from the bidirectional self-inactivatingtransfer vector pCCLsin.cPPT.Δ LNGFR.minCMV.hPGK.eGFP.Wpre and frompCCLsin.cPPT.hPGK.eGFP.Wpre can be generated and packaged by anintegrase-competent third-generation construct and pseudotyped by theVSV envelope.

Using vectors such as those above, and as described in Provasi, et al.,Nature Medicine, Vol. 18, No. 5, May 2012, it has been shown that ZFNspromoted the disruption of endogenous TCR β- and α-chain genes.Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR andexpanded with the addition of interleukin-7 (IL-7) and IL-15. Further,after lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1)antigen, the TCR-edited cells expressed new TCR at high levels (also asdescribed in Provasi, et al., Nature Medicine, Vol. 18, No. 5, May2012).

Example 32 Viral Transfer Vector with a Zinc Finger Nuclease Transgene(Prophetic)

Zinc finger nucleases (ZFNs) targeting the hF9mut locus and F9-targetingvectors can be prepared as described in Li, et al. Nature. 2011;475(7355):217-221. Such vectors have been shown to be successfully usedfor in vivo gene targeting in a neonatal mouse model of hemophilia B(HB). Systemic codelivery of the AAV vectors, encoding the ZFN pairtargeting the human F9 gene and a gene-targeting vector with arms ofhomology flanking a corrective cDNA cassette resulted in the correctionof a defective hF9 gene engineered into the mouse genome in the liversof such mice. Further, stable levels of human factor IX expressionsufficient to normalize clotting times was achieved.

Example 33 Viral Transfer Vector with a Meganuclease Transgene(Prophetic)

Any one of the viral vectors described herein, such as in the aboveExamples, may be used to produce a viral transfer vector with a geneediting transgene that encodes a meganuclease. Alternatively, and as anexample, the following describes a general methodology for producingsuch a viral transfer vector with a gene editing transgene. Themeganuclease may be any one of the meganucleases provided in U.S.Publication Nos. 20110033935 and 20130224863.

In some embodiments, particular viral genes are inactivated to preventreproduction of the virus. Preferably, in some embodiments, a virus isaltered so that it is capable only of delivery and maintenance within atarget cell, but does not retain the ability to replicate within thetarget cell or tissue. One or more DNA sequences encoding a meganucleasecan be introduced to the altered viral genome, so as to produce a viralgenome that acts like a vector. In some embodiments, the viral vector isa retroviral vector such as, but not limited to, the MFG or pLJ vectors.An MFG vector is a simplified Moloney murine leukemia virus vector(MoMLV) in which the DNA sequences encoding the pol and env proteins aredeleted to render it replication defective. A pll retroviral vector isalso a form of the MoMLV (see, e.g., Korman et al. (1987), Proc. Nat'lAcad. Sci., 84:2150-2154). In other embodiments, a recombinantadenovirus or adeno-associated virus can be used to produce a viralvector.

Example 34 Viral Transfer Vector with an Exon Skipping Transgene(Prophetic)

Any one of the viral vectors described herein, such as in the aboveExamples, may be used to produce a viral transfer vector with an exonskipping transgene. Alternatively, and as an example, the followingprovides a method for producing a viral transfer vector with a specificexon skipping transgene.

A three-plasmid transfection protocol can be used withpAAV(U7smOPT-SD23/BP22) and pAAV(U7smOPT-scr) plasmids for generation ofsingle-strand AAV1-U7ex235 and AAV1-U7scr; and scAAV-U7ex23 plasmid forself-complementary scAAV9-U7ex239. pAAV(U7smOPT-scr) plasmid can containthe non specific sequence GGTGTATTGCATGATATGT that does not match to anymurine cDNAs.

Use of a viral transfer vector produced according to the above, asdescribed in Le Hir et al., Molecular Therapy vol. 21 no. 8, 1551-1558August 2013 showed that such a vector can be used to restore dystrophin.However, the restoration decreased significantly between 3 and 12months, which was correlated with viral genome loss. Accordingly, thecompositions and methods provided herein can help maintain the effect ofsuch a treatment.

Example 35 Viral Transfer Vector with an Exon Skipping Transgene(Prophetic)

Clone U1#23 can be obtained by inverse PCR on the human U1 snRNA gene,with oligos mU1anti5 (5′-CGAAATTTCAGGTAAGCCGAGGTTATGAGATCTTGGGCCTCTGC-3′and mU1anti3 (5′-GAACTTTGCAGAGCCTCAAAATTAAATAGGGCAGGGGAGATACCATGATC-3′).The antisense-containing insert can be amplified from correspondingplasmid with oligos U1cas-up-NheI (5′-CTAGCTAGCGGTAAGGACCAGCTTCTTTG-3′)and U1cas-down-NheI (5′-CTAGCTAGCGGTTAGCGTACAGTCTAC-3′). The resultingfragment can be NheI-digested and cloned in the forward orientation ofthe pAAV2.1-CMV-EGFP plasmid.

AAV-U1#23 vector can be produced by triple transfection of 293 cells,purified by CsCl2 ultracentrifugation and titered by using bothreal-time PCR-based and dot-blot assays. The number of green-formingunits can be assessed by serial dilution on 293 cells. AAV vector can beproduced by the AAV TIGEM Vector Core.

Six-week-old mdx mice can be administered with 3-4×10¹² genome copies ofAAV vector via tail vein. Six and 12 weeks after virus administration,animals can be killed, and muscles from different districts can beharvested. EGFP analysis and dissections can be performed under afluorescent stereomicroscope (Leica MZ16FA).

Use of a viral transfer vector produced according to the above, asdescribed in Denti et al., 3758-3763, PNAS, Mar. 7, 2006, vol. 103, no.10, resulted in persistent exon skipping in mdx mice by tail veininjection. Systemic delivery of the vector resulted in effectivebody-wide colonization, significant recovery of functional properties invivo, and lower creatine kinase serum levels. The results suggest thatthere was a decrease in muscle wasting.

Example 36 Viral Transfer Vector with an Exon Skipping Transgene(Prophetic)

Different U7snRNA constructs specific to certain exons can beengineered, such as from U7smOPT-SD23/BP22 (modified murine U7snRNAgene). Antisense sequences targeting certain exons can be replaced byantisense sequences targeting exons of dystrophin mRNA that induce exonskipping as antisense oligonucleotides. Sequences can be inserted intoU7snRNA constructs. Resulting U7snRNA fragments can then be introducedeither in a lentiviral vector construct for further lentiviralproduction or into an AAV vector construct for AAV production.

Lentiviral vectors can be based on pRRLcPPT-hPGK-eGFP-WPRE constructswhere the hPGK-GFP cassette is removed and replaced with the U7snRNAconstruct. Lentiviral vectors can be generated by transfection into 293Tcells of a packaging construct, pCMVΔR8.74, a plasmid producing thevesicular stomatosis virus-G envelope (pMD.G) and the vector itself aspreviously described. Viral titers (infectious particles) can bedetermined by transduction of NIH3T3 cells with serial dilutions of thevector preparation in a 12-well plate. Seventy-two hours later, genomicDNA from transduced cells can be extracted using a genomic DNApurification kit (Qiagen, Crawley, UK). The infectious particles titer(infectious particle/ml) can be determined by quantitative real-time PCRas described elsewhere.

For subsequent AAV vector production, different U7snRNA fragments can beintroduced at the XbaI site of the pSMD2 AAV2 vector. AAV2/1 pseudotypedvectors can be prepared by cotransfection in 293 cells of pAAV2-U7snRNA,pXX6 encoding adenovirus helper functions and pAAV1pITRCO2 that containsthe AAV2 rep and AAV1 cap genes. Vector particles can be purified onIodixanol gradients from cell lysates obtained 48 hours aftertransfection and titers can be measured by quantitative real time PCR.

As described in Goyenvalle, et al. The American Society of Gene & CellTherapy, Vol. 20 No. 6, 1212-1221 June 2012, viral transfer vectors suchas those produced and encoding U7 small-nuclear RNAs with the abovemethods can induce efficient exon skipping both in vitro and in vivo.

Example 37 Viral Transfer Vector with an Exon Skipping Transgene(Prophetic)

An HSV transfer vector can prepared according the methods of U.S.Publication No. 20090186003 and Example 28 above except that themethodology can be altered so that the transgene is instead an exonskipping transgene. The exon skipping transgene may be any one of suchtransgenes as described herein or otherwise known in the art.

Example 38 Viral Transfer Vector with an Exon Skipping Transgene(Prophetic)

An HIV lentiviral transfer vector is prepared according the methods ofU.S. Publication No. 20150056696 and Example 26 above except that themethodology can be altered so that the transgene is instead an exonskipping transgene. The exon skipping transgene may be any one of suchtransgenes as described herein or otherwise known in the art.

Example 39 Viral Transfer Vector with a Gene Expression ModulatingTransgene (Prophetic)

Any one of the viral vectors described herein, such as in the aboveExamples, may be used to produce a viral transfer vector with a geneexpression modulating transgene. Alternatively, and as an example, thefollowing provides a method for producing a viral transfer vector with aspecific gene expression modulating transgene.

A viral transfer vector is produced according to the methods describedin Brown et al., Nat Med. 2006 May; 12(5):585-91. Briefly, a plasmid isconstructed using reverse transcription of RNA, quantitative PCRanalysis to quantify the concentration of mRNA, and GAPDH expression fornormalization. VSV-pseudotyped third-generation lentiviral vectors (LVs)are produced by transient four-plasmid cotransfection into 293T cellsand purified by ultracentrifugation. Vector particles can be measured byHIV-1 gag p24 antigen immunocapture.

As described in Brown et al., Nat Med. 2006 May; 12(5):585-91, suchlentiviral vectors encoding target sequences of endogenous miRNAs wereshown to result in the production of miRNAs that could segregate geneexpression in different tissues. Evidence of miRNA regulation wasprovided and demonstrates that such vectors may be used in therapeuticapplications.

Example 40 Viral Transfer Vector with a Gene Expression ModulatingTransgene (Prophetic)

To produce an AAV2/1 serotype vector encoding an miRNA-based hairpinagainst a gene (e.g., huntingtin gene; AAV2/1-miRNA-Htt), the cDNA forthe specified gene (e.g., human HTT), can be cloned into a shuttleplasmid containing the AAV2 inverted terminal repeats (ITRs) and a1.6-kb cytomegalovirus enhancer/chicken b-actin (CBA) promoter. Controlvectors can also be developed and contain either an empty vectorbackbone (e.g., AAV2/1-Null) or express a reporter such as enhancedgreen fluorescent protein under the control of the same promoter(AAV2/1-eGFP). Viral transfer vectors can be generated by triple-plasmidcotransfection of a cell line, such as human 293 cells, and therecombinant virions can then be column-purified as previously describedin Stanek et al., Human Gene Therapy. 2014; 25:461-474. The resultingtiter of AAV2/1-miRNA-Htt can then be determined using quantitative PCR.

Data generated using such viral transfer vectors, as described in Staneket al., Human Gene Therapy. 2014; 25:461-474, demonstrated thatAAV-mediated RNAi can be effective at transducing cells in the striatumand can partially reduce the levels of both wild-type and mutant Htt inthis region.

Example 41 Viral Transfer Vector with a Gene Expression ModulatingTransgene (Prophetic)

The CD81 gene can be amplified by reverse transcription. cDNA can be PCRamplified with appropriate primers. The forward primer can contain aBamHI (Biolabs, Allschwill, Switzerland) restriction site followed by a5′ CD81 cDNA-specific sequence; the reverse primer can contain a 3′ CD81cDNA-specific sequence, a 6 His-tag, a stop codon and an Xho I (Biolabs)restriction site. The PCR product can be digested and cloned intosimilar sites in pTK431. The pTK431 is a self-inactivating HIV-1 vectorwhich contains the entire tet-off-inducible system, the centralpolypurine tract (cPPT) and the woodchuck hepatitis viruspost-transcriptional regulatory element. Plasmids can be CsCl₂ purified.

Targets can be designed according to the CD81 mRNA sequence based onHannon's design criterion (katandin.cshl.org:9331/RNAi/html/rnai.html).Using the pSilencer 1.0-U6 (Ambion) as a template and a U6promoter-specific forward primer containing a restriction site, eachshRNA target can be added to the mouse U6 promoter by PCR. The PCRproduct can be digested, cloned into similar sites in pTK431 andsequenced to verify the integrity of each construct.

The vector plasmids, together with a packaging construct plasmid pΔNRFand the envelope plasmid pMDG-VSVG, can be cotransfected into HEK293Tcells to produce viral particles. The viral titres can be determined byp24 antigen measurements (KPL, Lausanne, Switzerland).

As shown in Bahi, et al. J. Neurochem. (2005) 92, 1243-1255,lentiviruses expressing short hairpin RNA (shRNA) targeted against CD81(Lenti-CD81-shRNAs) resulted in gene silencing after infection ofHEK293T cells in vitro. In addition, in vivo delivery ofLenti-CD81-shRNA resulted in silencing of endogenous CD81.

Example 42 Viral Transfer Vector with a Gene Expression ModulatingTransgene (Prophetic)

Any one of the viral vectors described herein, such as in the aboveExamples, may be used to produce a viral transfer vector with a geneexpression modulating transgene that encodes a RNAi agent. An example ofa RNAi agent that can be encoded by a gene expression modulatingtransgene as provided herein is described below.

An expression construct can include a promoter driving the expression ofthree or more individual shRNA species. The synthesis of small nuclearRNAs and transfer RNAs can be directed by RNA polymerase III (pol III)under the control of pol III-specific promoters. Because of therelatively high abundance of transcripts directed by these regulatoryelements, pol III promoters, including those derived from the U6 and H1genes, can be used to drive the expression of 1-x RNAi (see, e.g.,Domitrovich and Kunkel. Nucl. Acids Res. 31(9): 2344-52 (2003); Boden,et al. Nucl. Acids Res. 31(17): 5033-38 (2003a); and Kawasaki, et al.Nucleic Acids Res. 31(2): 700-7 (2003)). RNAi expression constructsusing the U6 promoter can comprise three RNAi agents targeting threedifferent regions of the HCV genome. Further examples of RNAi agentsthat may be encoded by a gene expression modulating transgene includeany one of the RNAi agents described herein.

Example 43 Viral Transfer Vector with a Gene Expression ModulatingTransgene (Prophetic)

Any one of the viral vectors described herein, such as in the aboveExamples, may be used to produce a viral transfer vector with a geneexpression modulating transgene that encodes a Serpinal RNAi agent, suchas one of such agents described in U.S. Patent Publication No.20140350071. A viral transfer vector with such a transgene can beproduced following similar methodology as provided herein or otherwiseknown in the art.

For example, adeno-associated virus (AAV) vectors may be used (Walsh etal., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No.5,436,146). The iRNA can be expressed as two separate, complementarysingle-stranded RNA molecules from a recombinant AAV vector having, forexample, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV)promoter. Suitable AAV vectors for expressing the dsRNA featured in theinvention, methods for constructing the recombinant AV vector, andmethods for delivering the vectors into target cells are described inSamulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al.(1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941;International Patent Application No. WO 94/13788; and InternationalPatent Application No. WO 93/24641, the entire disclosures of suchinformation are herein incorporated by reference.

Example 44 Establishing an Anti-Viral Transfer Vector AttenuatedResponse in a Subject (Prophetic)

Any one of the viral transfer vectors provided herein, such as any oneof the Examples, is administered concomitantly, such as simultaneouslyi.v., i.m., s.c. or i.p., with any one of the antigen-presenting celltargeted immunosuppressants as provided herein, such as any one of theExamples, that is also administered i.v., i.m., s.c. or i.p.,respectively. The administration occurs according to a protocol,including at least the frequency and dose of the viral transfer vectorand antigen-presenting cell targeted immunosuppressant, that establishesan anti-viral transfer vector attenuated response in the subject. Thesubject may be any one of the subjects described herein, such as onethat does not have a pre-existing immunity to the viral transfer vectoror one in which repeated administration of the viral transfer vector isdesired.

In some embodiments, when the anti-viral transfer vector attenuatedresponse is a T cell response against the viral transfer vector, theviral transfer vector is administered to the subject without anantigen-presenting cell targeted immunosuppressant prior to theconcomitant administration of the antigen-presenting cell targetedimmunosuppressant and viral transfer vector. In such embodiments, one ormore repeat doses of the viral transfer vector is administered to thesubject subsequent to both the concomitant administration and theadministration of the viral transfer vector prior thereto.

In some embodiments, when the anti-viral transfer vector attenuatedresponse is a B cell response against the viral transfer vector, thesubject is not administered the viral transfer vector prior to theconcomitant administration of the viral transfer vector andantigen-presenting cell targeted immunosuppressant. In such embodiments,one or more repeat doses of the viral transfer vector is administered tothe subject and each repeat dose is concomitantly administered with theantigen-presenting cell targeted immunosuppressant.

In other embodiments, when the anti-viral transfer vector attenuatedresponse is an anti-viral transfer vector antibody response, the subjectis not administered the viral transfer vector prior to the concomitantadministration of the viral transfer vector and antigen-presenting celltargeted immunosuppressant. In such embodiments, one or more repeatdoses of the viral transfer vector is administered to the subject andeach repeat dose is concomitantly administered with theantigen-presenting cell targeted immunosuppressant.

The method for determining the level of antibodies may be with the useof an ELISA assay. Assays for antigen-specific B cell or T cell recallresponses include, but are not limited to, ELISpot, intracellularcytokine staining, cell proliferation, and cytokine production assays.

In any one of the embodiments, the anti-viral transfer vector attenuatedresponse is evaluated after the concomitant administration of the viraltransfer vector and the antigen-presenting cell targetedimmunosuppressant.

In any one of the embodiments, a protocol for establishing theanti-viral transfer vector attenuated response may be determined. Insuch an embodiment, the protocol is determined in another subject, suchas a test subject. The protocol so determined can be used to treat othersubjects subjects in need of treatment with the viral transfer vector.

Example 45 Determining a Level of Pre-Existing Immunity in a SubjectPrior to Administration of a Viral Transfer Vector (Prophetic)

A sample, such as a blood sample, may be obtained from a subject that isin need of treatment with a viral transfer vector as provided herein,such as the viral transfer vector of any one of the viral transfervectors provided herein, such as in any one of the Examples.

With the sample from the subject, the level of antibodies, such asneutralizing antibodies or antigen recall responses of immune cells,such as T cells or B cells, can be determined. The method fordetermining the level of antibodies may be with the use of an ELISAassay. Assays for antigen-specific B cell or T cell recall responsesinclude, but are not limited to, ELISpot, intracellular cytokinestaining, cell proliferation, and cytokine production assays. The recallresponse can be assessed by contacting the sample with the viraltransfer vector or an antigen thereof. Alternatively, the recallresponse can also be assessed by taking the sample from the subjectafter administration of the viral transfer vector or an antigen thereofto the subject and then determining the level of antibodies or a B cellor T cell recall response that was generated.

In some embodiments, where the subject does not have a pre-existingimmunity to the viral transfer vector, determined by the measurement ofa level of anti-viral transfer vector antibodies in the subject (or a Bcell response), the subject is administered, i.v., i.m., s.c. or i.p.,any one of the viral transfer vectors provided herein, such as in anyone of the Examples, concomitantly, such as simultaneously, with any oneof the antigen-presenting cell targeted immunosuppressants providedherein, such as in any one of the Examples. The antigen-presenting celltargeted immunosuppressant is administered by the same route.

In other embodiments, where the subject does not have a pre-existingimmunity to the viral transfer vector, determined by the level of a Tcell response against the viral transfer vector in the subject, theantigen-presenting cell targeted immunosuppressant and viral transfervector are concomitantly, such as simultaneously, administered, i.v.,i.m., s.c. or i.p., to the subject after the subject is administered adose of the viral transfer vector without concomitant administration ofthe antigen-presenting cell targeted immunosuppressant.

In any one of the embodiments, one or more repeat doses of the viraltransfer vector is/are administered to the subject. These repeat dosesmay be concomitantly administered with the antigen-presenting celltargeted immunosuppressant.

Example 46 Escalating Transgene Expression of a Viral Transfer Vector ina Subject (Prophetic)

Any one of the viral transfer vectors provided herein, such as in anyone of the Examples, is administered concomitantly, such assimultaneously, i.v., i.m., s.c. or i.p., with any one of theantigen-presenting cell targeted immunosuppressants as provided herein,such as in any one of the Examples, according to a frequency and dosingthat escalates transgene expression (the transgene being delivered bythe viral transfer vector). This can be determined by measuringtransgene protein concentrations in various tissues or systems ofinterest in the subject. Whether or not transgene expression isescalated can be determined according to a method, such as thatdescribed in the Examples above. The administration occurs according tothe frequency and dose of the viral transfer vector andantigen-presenting cell targeted immunosuppressant, that escalatestransgene expression in the subject. The subject may be any one of thesubjects described herein, such as one that does not have a pre-existingimmunity to the viral transfer vector or one in which repeatedadministration of the viral transfer vector is desired.

In any one of the embodiments, the frequency and dose that achievesescalating transgene expression is determined in another subject, suchas a test subject. This can also be determined by measuring transgeneprotein concentrations in various tissues or systems of interest in theother subject, such as with a method as described above. If thefrequency and dose achieves escalated transgene expression, asdetermined by the measured transgene protein concentrations, in theother subject, the concomitant, such as simultaneous, administration ofthe viral transfer vector and antigen-presenting cell targetedimmunosuppressant according to the frequency and dose can be used totreat other subjects in need of treatment with the viral transfervector.

Example 47 Repeated, Concomitant Administration with Lower Doses(Prophetic)

As provided herein, a subject can be evaluated for the level of apre-existing immunity to any one of the viral transfer vectors providedherein, such as any one of the viral transfer vectors any one of theExamples. Alternatively, a clinician may evaluate a subject anddetermine whether or not, if administered the viral transfer vector, thesubject is expected to develop an anti-viral transfer vector immuneresponse if the viral transfer vector is repeatedly administered to thesubject. This determination may be made based on the likelihood that theviral transfer vector will produce such a result and may be based onsuch a result in other subjects, such as test subjects, informationabout the virus that was used to generate the viral transfer vector,information about the subject, etc. Generally, if the expectation isthat an anti-viral transfer vector immune response is the likely result,the clinician selects a certain dose of the viral transfer vector as aresult of the expectation. However, in light of the inventor's findings,a clinician may now select and use lower doses of the viral transfervector than would have been selected for the subject. Benefits of lowerdoses can include reduced toxicity associated with dosing of the viraltransfer vector, and reduction of other off-target effects.

Accordingly, any one of the subjects provided herein can be treated withrepeated, concomitant, such as simultaneous, administration of any oneof the viral transfer vectors provided herein and any one of theantigen-presenting cell targeted immunosuppressants provided hereinwhere the doses of the viral transfer vector are selected to be lessthan the dose of the viral transfer vector that would have been selectedfor the subject if the subject were expected to develop anti-viraltransfer vector immune responses due to repeated dosing of the viraltransfer vector. Each dose of the viral transfer vector of the repeated,concomitant administration may be less than what would have otherwisebeen selected.

1. A method comprising: establishing an anti-gene therapy viral transfervector attenuated response in a subject by concomitant administration ofan antigen-presenting cell targeted immunosuppressant and gene therapyviral transfer vector to the subject, wherein the subject does not havepre-existing immunity against the gene therapy viral transfer vector.2-6. (canceled)
 7. A method comprising: attenuating an anti-gene therapyviral transfer vector response, wherein the anti-gene therapy viraltransfer vector response is a T cell response, by first administering toa subject a gene therapy viral transfer vector without anantigen-presenting cell targeted immunosuppressant, and subsequentlyconcomitantly administering the gene therapy viral transfer vector andan antigen-presenting cell targeted immunosuppressant to the subject. 8.The method of claim 7, further comprising administering to the subjectone or more repeat doses of the viral transfer vector subsequent to theconcomitant administration of the viral transfer vector and theantigen-presenting cell targeted immunosuppressant to the subject. 9-16.(canceled)
 17. A method comprising: repeatedly, concomitantlyadministering to a subject an antigen-presenting cell targetedimmunosuppressant and gene therapy viral transfer vector, and selectingone or more doses of the gene therapy viral transfer vector to be lessthan the dose of the gene therapy viral transfer vector that would beselected for the subject if the subject were expected to developanti-gene therapy viral transfer vector immune responses due to therepeated administration of the gene therapy viral transfer vector.18-25. (canceled)
 26. A method comprising: inducing an entity topurchase or obtain an antigen-presenting cell targeted immunosuppressantalone or in combination with a gene therapy viral transfer vector bycommunicating to the entity that efficacious repeated gene therapy viraltransfer vector dosing is possible by concomitant administration of theantigen-presenting cell targeted immunosuppressant and gene therapyviral transfer vector to a subject. 27-38. (canceled)
 39. The method ofclaim 1, wherein the concomitant administration is simultaneousadministration.
 40. The method of claim 1, wherein the subject is one towhich the viral transfer vector has not been previously administered.41. The method of claim 1, wherein the viral transfer vector is aretroviral transfer vector, an adenoviral transfer vector, a lentiviraltransfer vector or an adeno-associated viral transfer vector. 42-46.(canceled)
 47. The method of claim 1, wherein the gene therapy transgeneof the viral transfer vector encodes a therapeutic protein. 48-71.(canceled)
 72. The method of claim 1, wherein the antigen-presentingcell targeted immunosuppressant comprises an erythrocyte-bindingtherapeutic. 73-75. (canceled)
 76. The method of claim 1, wherein theantigen-presenting cell targeted immunosuppressant comprises anegatively-charged particle. 77-80. (canceled)
 81. The method of claim1, wherein the antigen-presenting cell targeted immunosuppressantcomprises an apoptotic-body mimic and one or more viral transfer vectorantigens. 82-85. (canceled)
 86. The method of claim 1, wherein theantigen-presenting cell targeted immunosuppressant comprises syntheticnanocarriers comprising an immunosuppressant. 87-114. (canceled) 115.The method of claim 1, wherein the immunosuppressant is an inhibitor ofthe NF-kB pathway.
 116. The method of claim 1, wherein theimmunosuppressant is rapamycin.
 117. (canceled)